Download Review Molecular Biology in Arteriosclerosis Research

Review
Molecular Biology in
Arteriosclerosis Research
David L. Williams
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The topics discussed In this article illustrate how molecular biology will have a
dramatic Impact on arteriosclerosis research. DNA clones for a small number of
relevant proteins have been isolated, and studies are underway in numerous laboratories to extend these initial studies. The techniques of molecular biology will provide
major advances in our understanding of numerous proteins directly or indirectly
involved in the atherogenlc process. Cloning technology will solve the primary structures of many proteins that can not be purified In quantities sufficient for classical
methods of analysis. Studies of regulation will benefit from the availability of DNA
probes, the ability to generate site-directed antibodies, and the use of reverse genetics to Identify nucleic acid sequences involved in the regulation of gene expression.
Studies of gene structure and genetic polymorphisms will unravel the genetic basis
for defects In llpld and lipoprotein metabolism and should provide valuable reagents
for clinical screening and diagnosis. The reverse genetics approach will permit the
systematic analysis of structure-function relationships at the protein level In a manner
not previously possible. Each of these will contribute to our understanding of the
atherogenic process and should provide insight into ways of preventing and treating
arteriosclerosis. (Arteriosclerosis 5:213-227, May/June 1985)
he purpose of this article is to explore how molecT
ular biology can have an impact on research in
the broad field of arteriosclerosis. The past few years
in this area. In the very near future, clones for many
more proteins of importance to arteriosclerosis will
be isolated. In addition to the initial information from
cloning experiments regarding protein, mRNA, and
gene structure, these studies will form the foundation
for the analysis of important macromolecules at a
level of understanding not previously available. This
is true in terms of structure and structure-function
relationships as well as in terms of regulation. Furthermore, it is likely that molecular biology will contribute significantly to the clinical side of arteriosclerosis in diagnostics and ultimately in therapeutics.
The subsequent discussion aims to illustrate the
above points by proceeding from present knowledge
to the fantasy of what may be in the not-too-distant
future. First, as a general introduction, strategies for
the isolation of genes will be presented. Second, the
most immediate consequence that molecular biology will have on arteriosclerosis research, that is, the
unraveling of primary sequences of key proteins, will
be considered. Third, molecular genetics, including
aspects of gene structure, regulation, genetic polymorphisms, and reverse genetics will be discussed.
These topics represent the potential that arteriosclerosis research will realize from the application of the
techniques of molecular biology.
have seen the techniques of molecular biology applied to an increasingly wider range of problems directly or indirectly related to arteriosclerosis. The first
focus of research in this area has been the isolation
and characterization of complementary DNA (cDNA)
clones for the major apolipoproteins of the plasma
lipoproteins. The apolipoproteins were prime targets
for initial studies because they are abundant products of lipoprotein-producing tissues and the amino
acid sequences of many of these proteins are
known. While the current literature seems filled with
cloning papers, we only have scratched the surface
From the Department of Pharmacological Sciences, Health Sciences Center, State University of New York at Stony Brook, Stony
Brook, New York.
This work was supported by Grants AM 18171 and HL 32868
from the National Institutes of Health.
Address for reprints: David L. Williams, Department of Pharmacological Sciences, Hearth Sciences Center, State University of
New York at Stony Brook, Stony Brook, New York 11794.
Received November 2, 1984; revision accepted January 22,
1985.
213
ARTERIOSCLEROSIS
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VOL
5, No 3,
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Introns
kbp
mRNA
oligo dT
PDGF
RFLP
poly A
T
v-sis
1985
Isolation and Characterization of Genes
Abbreviations
A
arg
C
cDNA
c-myc
cys
dC
dG
DHFR
(ds)
EcoRI
ELH
exons
Q
HMG CoA reductase
MAY/JUNE
As an introduction, it is instructive to consider typical methods for the isolation and characterization of
genes.1 In most cases, the isolation of a gene first
requires the isolation of cDNA clones for the messenger RNA (mRNA) of interest. The isolation of a
specific cDNA clone involves the construction of an
appropriate cDNA library and the identification of the
specific cDNA within that library. The mRNA is first
purified from a tissue that makes the protein of interest. In the case of the plasma apolipoproteins, liver is
an appropriate source of the mRNA.
Figure 1 shows a typical scheme2 for library
construction in which cDNA is prepared by priming
the reverse transcription of mRNA with oligo deoxythymidylic acid (oligo dT) hybridized to the polyadenylic acid (poly A) at the 3' end of most mRNA
molecules. After first-strand synthesis and removal
of the template mRNA by alkaline hydrolysis, a second strand of cDNA is synthesized with DNA polymerase. In the example shown, second-strand synthesis is primed by a transient hairpin structure at the
3' end of the cDNA. The double-stranded (ds)cDNA
is treated with S1 nuclease to remove the hairpin
loop and single-stranded regions at the 5' end of the
adenlne
arginlne
cytoslne
complementary DNA
an oncogene
cystelne
deoxycytidyllc acid
deoxyguanyllc acid
dlhdrofolate reductase
double stranded
restriction endonuclease
egg-laying hormone
coding DNA segments
guanlne
3-hydroxy-3-methyl-glutaryl
coenzyme A reductase
noncodlng DNA segments
kllobase pairs
messenger RNA
ollgo deoxythymldylic acid
platelet-derived growth factor
restriction fragment length
polymorphism
polyadenyllc acid
thymlne
an oncogene
5'-
mRNA
- A A A B 3'
CMKJOWT)
AAA B
TTTn
( I ) Reverse tronscnptose
(2)Alkdi
T T T . 5'
DNA
Polymerase + 4 dNTPs.
5'
3'
G
3' ACGTC
GGjGACGTC
CC n C
r
G
CCjC
-CTGCAGCG
Select
Tc*. A p s
-GtaCGTCC"C-CTGCAIGGJS
cDNA
GGnGACGTCCCnC
G"
Transformation
host repairs gaps,
reconstruct* Pstl
vies
GGGLACGTC
CC.CTGCA
Figure 1. Construction of an oligo dT-primed cDNA library in pBR
322 with the G-C tailing procedure. Reprinted with permission from
Old and Primrose.2
MOLECULAR BIOLOGY AND ARTERIOSCLEROSIS
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first strand. The resultant blunt-ended, doublestranded cDNA is tailed with deoxycytidylic acid (dC)
residues at the 3' ends via reaction with terminal
transferase and dCTP. The dC-tailed cDNA is annealed with vector pBR 322 DNA that has been linearized by restriction endonuclease digestion at the
unique Pst I site and tailed with deoxyguanylic (dG)
acid residues.
As illustrated, the cDNA molecules are incorporated into the vector DNA via base pairing between the
oligo dC and the oligo dG tails. After transformation
of the host bacterium with this hybrid DNA molecule,
the DNA repair systems fill in the gaps and regenerate the Pst I restriction site; this permits researchers
to recover the inserted DNA from the vector by Pst I
digestion.3 Another feature of this scheme is that the
Pst I site lies within the vector gene encoding resistance to ampicillin. Thus, bacteria transformed with
vector molecules containing cDNA inserts will be resistant to tetracycline by virtue of the vector TcR gene
but sensitive to ampicillin, since the cDNA insert has
disrupted the ApR gene.3
A cDNA library formed as illustrated (Figure 1) or
with a variety of alternate strategies generally consists of 103—10s recombinant DNA molecules, each
present in a different bacterium. The frequency with
which a specific cDNA clone occurs in the library is
approximately proportional to the abundance of the
specific mRNA within the mRNA population. Since a
typical mammalian cell may contain 2 to 5 x 105
mRNA molecules, a very large library must be constructed to ensure the inclusion of low abundance
mRNAs that are present in only a few copies per cell.
However, a small cDNA library may suffice for the
isolation of cDNA clones for high abundance
mRNAs.
Once the library is constructed, the specific cDNA
clone of interest must be identified. Table 1 lists a
215
Williams
number of cDNA clones recently isolated and the
strategies used to identify the particular clones. The
avian very low density lipoprotein (VLDL) apoprotein
(apo) II cDNA clone was the first apolipoprotein
clone isolated.45 In this case, the cDNA was prepared from highly purified apo II mRNA, thereby simplifying the identification of the cDNA clone. The apo
II cDNA clone was also identified in unfractionated
liver cDNA libraries by hybridization with a kinetically
fractionated cDNA probe6 and by a plus-minus
screening procedure.7 In the latter case, the library
was screened in parallel with radiolabeled cDNA prepared to mRNA from livers of estrogen-stimulated
chickens (plus probe) and with cDNA prepared to
mRNA from livers of nonstimulated chickens (minus
probe). Since apo II mRNA is only synthesized in
response to estrogen, the double screening with plus
and minus probes readily identified cDNA clones
corresponding to estrogen-induced mRNAs. Nucleotide sequence analysis confirmed the identity of the
apo II clone isolated in this manner.7
Screening of a cDNA library with a kinetically fractionated cDNA probe was also used to identify putative clones to rat apo A IV.19 The identity of the apo A
IV clone was confirmed by a hybrid selection translation procedure in which the cDNA clone was used to
purify the mRNA via hybridization. The purified
mRNA was then translated in vitro, and the translation product was identified as apo A IV. Similar
schemes involving partial mRNA purification and hybrid selection translation have been used to identify
cDNA clones to mouse apo A-l,12 mouse apo E,18
and rat apo E16 mRNAs (Table 1). Human apo E
cDNA clones were identified by screening a cDNA
library with a probe prepared from the rat apo E
cDNA.'5
An approach which has proved to be widely applicable is the use of oligonucleotide probes to identify
Table 1. cDNA Clones
Protein
Apo II
Apo II
Apo II
Apo A-l
Apo A-l
ApoE
ApoE
ApoE
ApoE
ApoE
Apo A-IV
Apo A-ll
Apo C-ll
ApoC-l
Apo C-lll
HMG CoA reductase
LDL receptor
Species
Chicken
Chicken
Chicken
Human
Mouse
Human
Human
Rat
Rat
Mouse
Rat
Human
Human
Human
Human
Chinese hamster
Cow
Method
mRNA purification
cDNA enrichment
Plus/minus screening
Oligonucleotide probe
Hybrid selection translation
Oligonucleotide probe
Homology with rat probe
Hybrid selection translation
Expression library
Hybrid selection translation
cDNA enrichment-hybrid selection translation
Oligonucleotide probe
Oligonucleotide probe
Oligonucleotide probe
Via linkage to apo A-l gene
Hybrid selection translation
Oligonucleotide probe
Reference
4,5
6
7
8-11
12
13,14
15
16
17
18
19
20
20-22
23
20,24
25
26
ARTERIOSCLEROSIS
216
Table 2.
VOL
5, No 3,
MAY/JUNE
1985
Apo A-l Oligonucleotide Probe
Protein or nucleic acid
Sequence
Amino acid sequence
105
gin
106
lys
107
lys
108
trp
109
gin
Potential mRNA sequence
5'
CAG
A
AAG
A
AAG
A
UGG
CAG
A
Mixed oligonucleotide probe
3'
TC
T
TTC
T
TTC
T
ACC
GTC
T
Actual sequence of apo A-l mRNA
5'
CAG
AAG
AAG
UGG
CAG
3'
The table shows the amino acid sequence of apo A-l for residues 105-109, the potential codons that
could code for each amino acid, the mixed oligonucleotide probe prepared by Breslow et al.,9 and the
actual sequence of apo A-l mRNA as determined by Breslow et al. from the apo A-l cDNA.
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
specific cDNAs. When the complete or partial amino
acid sequence of a protein is known, sequence regions encoded with minimal redundancy in the genetic code can often be identified, and an appropriate oligonucleotide probe to this region can be
chemically synthesized. Table 2 shows an example
of this approach used by Breslow et al.9 to identify
apo A-l cDNA clones. The amino acid sequence of
reidues 105-109 is encoded by a nucleotide sequence that is redundant at only four positions. A
mixed oligonucleotide probe that included both C
and T at the redundant positions was prepared, radiolabeled, and used to screen a human liver cDNA
library. Although only one of the 16 probes in the
mixture is a perfect match to the apo A-l cDNA sequence (Table 2), the hybridization was sufficiently
specific to identify putative apo A-l clones that were
confirmed by nucleotide sequence analysis.9
As shown in Table 1, this approach has been used
to identify cDNA clones to a variety of apolipoproteins for which the complete amino acid sequences
were known. This approach has also been used to
obtain cDNA clones to the bovine LDL receptor.26 In
this case, oligonucleotide probes were prepared on
the basis of a very limited amount of protein sequence data obtained by microsequencing of CNBr
fragments. The bovine cDNA subsequently was
used to identify a human LDL receptor genomic
clone, which in turn was used to isolate the cDNA for
the human LDL receptor.27 The full length cDNA
clone defines the entire primary amino acid structure
of the human LDL receptor.27 Limited microsequencing and oligonucleotide synthesis will clearly play a
major role in the isolation of cDNA clones to a variety
of low abundance proteins which are difficult or impossible to purify in amounts sufficient for classical
methods of analysis.
Table 1 also lists the identification of a rat apo E
clone via immunological screening of an expression
library.1728 In this case, cDNA fragments were
cloned into a bacteriophage lambda vector, and
protein fragments encoded by the cDNA were expressed under control of a regulated lambda gene.
The expressed apo E determinants were detected
with an antibody to apo E. Similar schemes for the
construction of expression libraries in plasmid vectors have been described.29 Expression libraries
should prove useful for the isolation of cDNA clones
to proteins for which high quality polyclonal antibodies are available.
A cDNA clone can serve as a reagent for the identification of DNA sequences within a genomic DNA
library. Since the mammalian genome is very large
(approximately 3 x 109 base pairs), genomic libraries are constructed with large fragments of DNA so
that the entire genome is represented in a library of
manageable size. This usually involves the use of
bacteriophage lambda as a cloning vector since very
large DNA fragments can be propagated in this
vector.
Figure 2 shows a typical scheme30 in which genomic DNA is partially digested with restriction endonucleases to produce fragments of approximately
20 kilobase pairs (kbp). After methylation to block
Eco Rl sites in the DNA and ligation to synthetic Eco
Rl linker molecules, the DNA fragments are annealed and ligated to a lambda vector which has
cohesive Eco Rl ends. The recombinant DNA molecules are packaged into phage particles and amplified on the appropriate bacterial host.
Since the DNA fragments are 20 kbp and the typical mammalian genome contains 2 to 3 x 106 kbp,
approximately 3 x 10s phage particles must be
screened to detect a gene present in one copy per
genome. For this purpose, phage and host bacteria
are plated as a lawn on an agar plate, and the plate is
incubated to permit plaques of lysed cells to develop.
The phage DNA is transferred to a nitrocellulose filter
by placing the filter on the surface of the plate. This
filter is then hybridized with a radiolabeled DNA
probe prepared from the appropriate cDNA clone,
and the radioactive signal is detected by autoradiography. The region of the original plate containing the
hybridizing DNA is recovered, and the phage carrying the desired DNA is purified by additional rounds
of plating and screening with the cDNA probe.
The initial cDNA clone usually provides the probe
for the detection of the gene in a homologous genomic library. However, nucleotide sequence homologies among species are often sufficient to permit
MOLECULAR BIOLOGY AND ARTERIOSCLEROSIS
Williams
217
X Charon 4A DNA
(replacement vector)
High molecular weight eukaryotic
DNA(>IOOkb)
Cleave with a
muture ofMwm and Mi
(very partial digest)
Sue froctionote
njrz/Fu. -20 kb
EcoRl methylose to
block EcoRl sites
Me
Me
Me
Me
Blunt end ligolion with
firoRI linker molecules
Me
Me
i
I
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Me
Me
internal fragments
Me
size
fractionate
to remove
internal
fragments
Me
i
Me
Me
anneal fcoRI cohesive
ends, ligote
Me
Me
Me
Me
Me
Me
Me
Me
m vitro packaging
phage particle
1
amplification and screening
Figure 2. Construction of a genomic DNA library in bacteriophage lambda Charon 4A. Reprinted with permission from Old and Primrose.2
the isolation of genes via cross-species hybridization. For example, we have recently isolated the apo
E gene from an African green monkey library by
using the human apo E cDNA clone prepared by
Breslow et al.13 (S Mungal, GS Shelness, T Newman, and DL Williams, unpublished data). When the
protein in question has been highly conserved during
evolution, genes may be identified with heterologous
probes from species much more divergent than this
example.
The isolated gene provides structural information
as well as specific probes and reagents for additional
studies. Generally, the first order of business is the
structural analysis of the gene by restriction endonuclease mapping and nucleotide sequencing. For the
list in Table 1, genes now have been isolated for
avian apo II, 3 1 3 2 human apo A-l 3 3 3 4 human apo C-
III,24 human apo E,35 human apo C-ll,21 human LDL
receptor,27 and Chinese hamster HMG Co A reductase.36 These genes are at various stages of analysis
in a number of laboratories. Nucleotide sequences
for the avian apo II gene37 and the human apo A-l
gene33-M have been reported. Gene organization will
be considered below.
Protein Structure
Primary Sequence
The immediate consequence of the isolation of a
cDNA clone is the availability of the amino acid sequence deduced from the nucleotide sequence of
the cDNA. Table 3 shows a partial list of proteins that
have been or are likely to be the initial targets for
cloning studies by investigators interested in arterio-
218
ARTERIOSCLEROSIS
VOL
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sclerosis. The proteins marked with an asterisk have
been sequenced by classical methods of protein
chemistry. In these cases, the cDNA clones provide
confirmatory data and serve to correct errors in the
protein data. In addition, the cDNA will yield the sequence of the protein presegment and the prosegment if these have not been determined by protein
sequencing of cell-free translation products. For
most of the proteins listed in Table 3, however, amino acid sequence data is not available, and cDNA
clones will be the most direct way to obtain this information when the protein can not easily be purified in
amounts sufficient for classical analysis.
Apo B is a good example of an important protein
that has baffled and frustrated protein chemists for
more than 15 years. Although apo B is present at
high concentrations in the blood, is relatively easy to
purify, and has been the subject of intensive investigation, very little is known about the structure or sequence of this protein. Much of the difficulty lies in the
insolubility of apo B, its tendency to aggregate, and
Table 3.
Initial Targets for Cloning
Category
Cloning target
Apolipoproteins
Apo B 100
Apo B48
Apo A-l*
Apo A-ll*
Apo A-IV
Apo C-l*
Apo C-M*
Apo C-lll*
Apo D
Apo E*
Apo F
Lpa determinant
Enzymes
Lecithin-cholesterol acyltransferase
(LCAT)
Fatty acyl-CoA:cholesterol acyltransferase (ACAT)
Peripheral lipoprotein lipases
Hepatic triglyceride lipase
HMG CoA reductase
HMG CoA synthase
Cholesterol ester hydrolases
Fatty acid and phospholipid synthetic enzymes
Cholesterol 7-alpha hydroxylase
Transport and
transfer proteins
Cholesterol ester transfer protein
Phospholipid transfer proteins
Retinol binding protein
Receptors for
Apo B/E (LDL)
Beta VLDL
Acetyl LDL
Apo E
HDL
Platelet-derived growth factor
Other factors regulating arterial
cell growth
*An asterisk indicates that the protein has been
sequenced by classical methods of protein chemistry.
5, No 3,
MAY/JUNE
1985
its susceptibility to proteolysis.38 As a result, even
such a fundamental property as the molecular weight
of apo B has remained controversial. Several research groups are now attempting to isolate cDNA
clones for apo B. Limited amino acid sequence data
obtained by one group have been used to construct
oligonucleotide probes to identify apo B clones.39
Several putative apo B clones were identified. An
alternative approach is the use of expression libraries that may permit apo B clones to be identified with
immunological techniques. In either case, we can
anticipate that the structure of this difficult protein will
be elucidated by these techniques in the not too distant future.
An example of an important protein for which the
amino acid sequence was deduced from the nucleotide sequence is the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) reductase. HMG
CoA reductase has been the subject of considerable
investigation because of its key role in cholesterol
biosynthesis and its dramatic regulation.40 However,
HMG CoA reductase is a very low abundance enzyme that has been difficult to purify and characterize. Chin et al.25 isolated a cDNA clone for HMG CoA
reductase from a cDNA library prepared from UT-1
cells, a clone of Chinese hamster ovary cells that
overproduces the enzyme and its mRNA. The initial
cDNA clone was used to identify additional cDNA41
and genomic36 clones that define the entire protein
and mRNA sequence and the gene sequence in the
region of transcription initiation. Analysis of the derived protein sequence has permitted the formulation
of an experimentally testable structural model for the
insertion of HMG CoA reductase in the endoplasmic
reticulum membrane / 2
Analyses of the mRNA and 5' flanking sequences
of the gene have identified sequences potentially
important for transcription initiation and the control of
gene expression.36 Thus, considerable new information about the structure and regulation of HMG CoA
reductase at both the protein and nucleic acid level
has been obtained primarily with the use of recombinant DNA techniques. Considerable progress also
has been made in the isolation and analysis of cDNA
clones for the bovine and human LDL receptor.2627
The rapid progress made in elucidating the structures of the LDL receptor and HMG CoA reductase
illustrates the potential of molecular biology for the
analysis of proteins relevant to arteriosclerosis.
Protein Variants
The availability of cDNA clones permits the analysis of protein variants at a level of understanding not
possible with biochemical techniques. Genetic variants have been recognized for apo E, apo A-l, apo
A-IV, apo B, and apo C-ll by an analysis of protein
charge or immunological properties.35 Elucidation of
the nucleotide sequences of the wild type and variant
alleles should explain the basis for the variants and
may contribute to our understanding of structurefunction relationships in these proteins.
MOLECULAR BIOLOGY AND ARTERIOSCLEROSIS
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Apo E provides an interesting example in which
variants have been studied at both the protein and
nucleic acid levels. It has long been known that apo E
exhibits charge heterogeneity upon isoelectricfocusing.43 High resolution, two-dimensional gel analysis
demonstrated six human apo E phenotypes that possibly resulted from three common alleles (e4, e3,
and e2) at a single genetic locus.44 The e3 allele was
designated as the wild type because it occurs with
the highest frequency. As shown in Table 4, the three
alleles differ by arginine (arg)-cysteine (cys) interchanges at residues 112 (e3 cys) and 158 (e3
arg).45^7 Subsequent analysis of the nucleotide sequence of the e3 allele showed that the codons for
112 (e3 TGC) and 158 (e3 CGC) could undergo
single base changes to account for the amino acid
differences in the e2 and e4 alleles.13 This also was
the case with an e2* variant that has cysteine instead
of arginine at position 145. Nucleotide sequencing of
the e4 allele has confirmed this interpretation.48
Thus, the charge differences between the alleles
have been explained both at the level of the amino
acid sequence and at the level of the nucleotide substitutions in the gene. This example of allelic variation also provides structure-function information
about the protein since the e2 allele binds poorly to
apo B/E receptors or hepatic apo E receptors.4649
Arginine 158 appears to reside within the receptor
binding domain or to influence receptor interactions
at another site on the apo E molecule. The physiological significance is that the e2 allele is strongly
associated with Type III hyperlipoproteinemia in
which patients show a reduced clearance of remnant
particles presumably due to the inefficient interaction
of the altered apo E with hepatic receptors.50"52
Nucleotide sequence analysis of other apo E
cDNA clones has provided evidence for additional
sites of variation in the gene and in the protein.35 In
some cases, base changes in the DNA are silent,
i.e., amino acid substitutions do not result. In other
Table 4.
Apo E Alleles and Nucleotide Substitutions
Amino acid
Amino acid
112
codon
158
codon
e3
CYS
TGC
ARG
CGC
e2
CYS
TGC
CYS
TGC
e4
ARG
CGC
ARG
CGC
Allele
The table shows the amino acids at positions 112 and
158 of the three common apo E alleles as determined by
amino acid sequence analysis (see references 45-47).
The codons specifying these amino acids are given in
terms of the DNA bases. The codons for the e3 and E4
alleles have been determined by nucleotide sequence
analysis of cDNA or genomic clones (see references 13,
15,35,48), which shows that the amino acid substitution is
due to a point mutation. The indicated nucleotide substitution in the E2 allele could account for the arg-cys interchange at residue 158, but the nucleotide sequence of the
E2 DNA has not yet been determined.
Williams
219
cases, there are amino acid substitutions that do not
involve charged residues and would not be detected
by isoelectric focusing. The analysis at the nucleotide sequence level is clearly more informative with
regard to genetic variation in apo E than is analysis at
the protein level. In general, this will be the case for
most proteins. In addition, nucleotide sequence
analysis is easier and faster than amino acid sequence analysis.
Antibodies to Synthetic Peptldes
Another consequence of the availability of the nucleotide sequence of a cDNA and the derived amino
acid sequence is the potential for generating antibodies to defined regions of the protein. It is now well
recognized that synthetic peptides can elicit antibodies that react with high frequency with the intact
native protein.53"55 Both polyclonal and monoclonal
antipeptide antibodies have been studied. As a result, it is possible to engineer antipeptide antibodies
as probes for specific regions of the protein. This
approach generally involves a hydrophilicity analysis
of the protein sequence to select regions most likely
to be antigenic.56 Appropriate oligopeptides are synthesized by established methods and used to elicit
antibodies by conventional procedures. Antibodies
to a variety of viral antigens have been prepared in
this fashion.53"55 Antipeptide antibodies to the LDL
receptor have been used to determine the orientation of the receptor in the plasma membrane.57 Antipeptide antibodies also have been used to analyze
the domain structure of HMG CoA reductase.42 This
approach should be applicable to almost any protein
for which the sequence is known or can be derived
from the cDNA.
Molecular Genetics
Gene Structure and Organization
The DNA sequences encoding most eucaryotic
proteins occur in multiple segments or exons which
are separated by noncoding DNA segments called
introns or intervening sequence DNA. Several strategies may be used to determine the arrangement of
exons and introns in the DNA and the organization of
exon sequences in the mature messenger RNA
(mRNA). The ultimate goal is to obtain the sequence
of the gene and the mRNA in order to define the
organization at the nucleotide level.
Figure 3 shows the organization of the apo II gene
as an example. Apo II is a small apolipoprotein of 82
amino acid residues that is synthesized in the avian
liver in response to estrogenic hormones.58 Apo II is
secreted from the liver on VLDL particles and functions in the transport of VLDL to the oocyte for egg
yolk formation. Mature apo II as found in VLDL is
illustrated at the bottom of Figure 3, while the preapo II polypeptide which includes the 24 amino acid
signal peptide is diagrammed above mature apo
II 37.59.60 A p o n m RNA of 659 nucleotides includes
ARTERIOSCLEROSIS
220
VOL
5, No 3,
MAY/JUNE
1985
abundance
5%
15%
80%
CCCCTCAC|TATATTA|QTTCTQ|CATAAATlQCCAOTOTCTCAQATOAQCATCA^CCTCAQCTTC»QCCTOQa
-50
-40
-SO
-20
-11
1
7
transcription
start
sites
2907
basepalrs
apo II
mRNA
669
bases
5' M Gppp-l
80 f
T
318
codo
for
NH2 terminus
of mature apo II
start
NH2-C
24
amlno
aclda
signal peptlda
261
stop
D-COOH
mature
plasma
apo II-VLDL
82 amlno acids
NHj-C
82 amlno acids
Organization of the avian apo II gene. Details are given in the text.
the 318 nucleotides that code for pre-apo II, a 5'
noncoding region of 80 nucleotides, and a 3' noncoding region of 261 nucleotides.3761
Also illustrated are the 3' polyadenylic acid track
and the 5'cap structure which are not encoded in the
DNA. As shown above apo II mRNA, the apo II gene
is composed of four exons (I-IV) and three introns
(A,B, and C) of the indicated sizes.37 After synthesis
of a primary transcript that includes all exons and
introns, the precursor mRNA is processed to remove
the intron sequences and splice the exon sequences
as illustrated. Note that exon I represents 5' untranslated sequence in the mRNA. Exon II encodes additional 5' untranslated sequence, the start codon for
translation, and most of the signal peptide for secretion of apo II. Exon III encodes the remainder of the
signal peptide and the amino terminal region of the
mature protein. Exon IV encodes the carboxyl region
of the protein and the entire 3' noncoding region of
the mRNA. The alpha helical regions of apo II believed to be important for lipid interactions5962 are
encoded mainly in exon IV.
It is striking that the overall organization of the
avian apo II gene is very similar to the organization of
the human apo A-l, apo C-III, and apo E genes.35
Each gene has a small 5' exon representing 5' untranslated sequence. In each case, the start codon
for translation and most of the signal peptide are
encoded by exon II, the amino terminal region of the
mature protein is encoded by exon III, and the carboxyl region and 3' untranslated region are encoded
by exon IV. Similarly, the amino acid sequence regions, which can form an amphipathic helix, are encoded by exon IV. These similarities may indicate a
common ancestral gene for avian apo II and the
mammalian apolipoproteins.
Gene Regulation
The entire nucleotide sequence of the apo II gene
and apo II cDNA have been determined.37 This information enables researchers to approach problems
of gene regulation at both the transcriptional and
translational levels. As an example, Figure 4 shows
the accumulation of apo II mRNA in the avian liver
after the administration of estradiol. Apo II mRNA
was measured with a DNA-excess solution hybridization assay employing a single-stranded probe.63
ell
Figure 3.
o
16,000
or
E 12,000
o
Q.
8,000
cn
riolecu
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>CO0H
primary
translation
product
preapo II
106
•mlno acids
4,000
A
12
24
36
48
60
72
Time After Estradiol (hours)
Figure 4. Estrogen-stimulated accumulation of apo II
mRNA. White leghorn roosters (0.4 kg to 0.6 kg) were
treated with estradiol (50 mg/kg). Liver RNA was prepared
at the indicated times. Apo II mRNA was measured with a
DNA-excess solution hybridization assay. Bracket indicates one SEM for measurements with three animals at
each point.
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Apo II mRNA accumulates to a level of 15,000 to
20,000 molecules/cell at which point it represents
about 10% of the total liver mRNA. This massive
accumulation of apo II mRNA results from estrogenstimulated transcription of the gene and hormone
action that stabilizes the mRNA against cytoplasmic
degradation.4"6 The stabilization of apo II mRNA presumably involves mRNA sequences in the 5' or 3'
untranslated regions. The availability of the nucleotide sequence and the appropriate cDNA clones permits researchers to ask which mRNA sequences are
necessary for stabilization. Similarly, at the DNA level they can ask which sequences are required for
transcription and hormone regulation.
Recent studies37-61 indicate that transcription is initiated at three sites on the apo II gene with different
frequencies as illustrated at the top of Figure 3. The
significance of multiple initiation sites and the reason
why each site is used with a different frequency are
not known. The nucleotide sequence adjacent to the
initiation sites includes two potential control sequences (boxed sequences) that are believed to be
important as promotor sequences in other genes.64
Studies are in progress in several laboratories to
define the function of these sequences and to identify gene sequences that interact with hormone receptors and are necessary for hormone-regulated
expression.
Similar studies with mammalian apolipoprotein
genes and a variety of genes relevant to arteriosclerosis are clearly of interest and will be pursued as
these genes become available. Problems of interest
are likely to include the effects of hormones and
metabolic intermediates on the expression of apolipoprotein genes, lipoprotein receptor genes, enzymes such as HMG CoA reductase, and a variety of
other enzymes involved in lipid metabolism. Similarly, researchers can view endothelial and smooth
muscle cell growth regulation in the arterial wall as
problems in gene regulation that can be approached
in a similar way. It is thought that platelet-derived
growth factor (PDGF) plays a significant role in stimulating smooth muscle cell growth in the atherosclerotic lesion.65 Recent studies indicate that PDGF is
highly homologous to the v-sis oncogene carried by
the simian sarcoma virus 6667 and can act in concert
with other factors to elicit transformation of fibroblasts.68 PDGF stimulation of cultured cells also results in the enhanced expression of another oncogene, c-myc.69 These data suggest that growth
stimulation in the arterial lesion may share common
pathways and cellular mediators with one type of
oncogenesis. One consequence of these findings is
a merging of the interests of investigators who work
in arteriosclerosis and those who do cancer research. An understanding of key steps in growth regulation at the molecular level will probably reveal
potential targets for pharmacological intervention in
arteriosclerosis as well as in neoplasia.
Another aspect of gene regulation is the tissuespecific expression of genes that is characteristic of
Williams
221
differentiated cells. What factors, for example, determine that apolipoprotein genes will be expressed in
the liver and small intestine but not in peripheral tissues? Similarly, the recent finding that apo E is synthesized in peripheral tissues70 and reticuloendothelial cells71 as well as in the liver raises the question of
how this gene is regulated in different cell types. In
recent studies, we have determined that the quantitative expression of the apo E gene in various tissues
of the nonhuman primate ranges over at least two
orders of magnitude.72 It is of interest to learn the
functional significance of these differences and the
mechanisms through which they are established.
Similar questions may be asked about a variety of
genes that show tissue-specific expression and are
relevant to arteriosclerosis.
In addition to the analysis of gene expression in
tissues or cell culture, DNA probes may be used as
histological reagents to examine in vivo gene expression at the cellular level. Figure 5 shows an example from work by McAllister et al.73 in which a
radiolabeled cDNA for the egg-laying hormone
(ELH) of the marine snail Aplysiawas used to identify
the neurons that synthesize this hormone. Figure 5 A
shows a section through the abdominal ganglion
which has been hybridized with 1 2 5 I-CDNA to ELH
and exposed by autoradiography. The dark staining
of the cell clusters in the upper left and right of the
ganglion represents intense hybridization of the
probe to ELH mRNA in the bag cell neurons. Figures
5 B and C are higher power magnifications of sections showing that the hybridization is restricted to
the bag cells. In Figure 5 C, the localization of grains
to the cell cytoplasm is evident. Figure 5 D shows
localization of the ELH peptide to the bag cells via
immunocytochemical staining with anti-ELH antibody. Note that the immunocytochemical technique
identified the protein product of the ELH gene while
the in situ hybridization technique identified the
mRNA for ELH. Thus, the immunocytochemical approach localized cells that contain the protein, while
the hybridization procedure localized cells that synthesize the protein. This distinction may be particularly important when attempting to identify cell types
that synthesize proteins normally present in the extracellular fluid or those taken up by cells, as is the
case with lipoproteins. Similar approaches could be
used to identify, within tissues, cell types that synthesize proteins such as apo E, lipoprotein lipase, or
LCAT.
Restriction Fragment Length Polymorphisms
A restriction fragment length polymorphism
(RFLP) refers to an altered pattern of gene fragments observed when genomic DNA from different
individuals is digested with a restriction endonuclease. Since a restriction endonuclease has a
specificity for a defined recognition sequence in the
DNA, an altered pattern of cleavage could be due to
as simple a change as a point mutation or to a com-
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Figure 5. In situ hybridization and immunocytochemistry to localize cells expressing the egg laying
hormone (ELH) in Aplysia. A. The abdominal ganglion from an adult animal was dissected, fixed with
Bouin's stain, sectioned, and hybridized with an 125l-labeled DNA probe for ELH mRNA. The bag cells
comprise the topmost rounded clusters of neurons on both sides of the ganglion. The photomicrograph shows hybridization to cells in both bag-cell clusters as well as to a single isolated neuron in the
upper left center of the ganglion. The hybridization is seen as the darkened areas due to the intense
concentration of silver grains on the autoradiograph. x 15. B. Hybridization to a section through a
single bag-cell cluster. Neurons are stained with methylene blue, x 58. C. A higher magnification (x
368) of bag-cell neurons after in situ hybridization. The deposition of silver grains over the cytoplasm of
nerve cell bodies can be seen. D. Immunohistochemical staining with anti-ELH antibody followed by
a second antibody coupled to peroxidase. The ELH-containing cells in the bag-cell cluster show darker
staining, x 230. Reprinted with permission from reference 73 (copyright 1983 by the American
Association for the Advancement of Science).
plex change such as a deletion, insertion, or rearrangement in the DNA.74 A typical analysis for
RFLP involves digestion of isolated DNA with a specific restriction enzyme, electrophoresis of the DNA
fragments on an agarose gel, blotting of the DNA
fragments in the gel to nitrocellulose paper by the
Southern transfer method,75 hybridization of the blot
with a radiolabeled cDNA or genomic DNA probe,
and autoradiography of the blot to detect the gene
fragments recognized by the probe. Genetic polymorphisms are useful markers for the classification
of specific phenotypes or for genetic linkage studies.
In some instances, a polymorphism can be instructive as to the basis for a metabolic disorder.
Figure 6 shows an example of an apo A-l gene
analysis carried out by Karathanasis et al.76 using
DNA from two patients with a familial deficiency of
plasma apo A-l and apo C-lll. The autoradiograph
shows that digestion of DNA from a normal individual
with the enzyme Eco Rl yields a 13 kbp fragment
detected with an apo A-l cDNA probe (Lane A). In
contrast, the patients have a 6.5 kbp Eco Rl fragment
instead of the 13 kbp fragment, indicating an alteration in the DNA in or near the apo A-l gene (Lanes F
and G). Since the patients have only the 6.5 kbp
fragment, they must be homozygous for this altered
apo A-l allele. Note that the mother (Lane D), father
(Lane C), and brother (Lane E) of the patients have
both fragments, indicating that they are heterozygous for the normal and mutant alleles. The patients
also showed a 0.95 kbp fragment in comparison to
the wild type, 2.2 kbp fragment when the DNA was
digested with the enzyme Pst I.76 These data suggested that the altered apo A-l gene might be the
basis for the lack of plasma apo A-l, but additional
studies were needed to define the mutant gene.
Figure 7 shows schematic representations of the
normal and mutant apo A-l genes and the cDNA
MOLECULAR BIOLOGY AND ARTERIOSCLEROSIS
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B ' C D E
F
223
Williams
G H
I
J K
23.1
9.4
6.5
4.3
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2.3
2.0
Figure 6. Restriction length polymorphism of the human apo A-l gene. The gel shows hybridization of an apo A-l cDNA probe to genomic DNA which has been digested with Eco Rl. The
DNA samples are from a normal individual (Lane A) and the apo A-l-deficient probands (Lanes F
and G). Other samples are from the maternal grandfather (Lane B), father (Lane C), mother
(Lane D), and brother (Lane E) of the probands. In addition, the son (Lane H) and daughter
(Lane I) of Proband G, and the son (Lane J) and daughter (Lane K) of Proband F are shown.
Reprinted with permission from Nature 301, 718-720 (1983); copyright 1983 Macmillan Journals Ltd.
Pi
Ri
I
1
I
II
IV
III
P2
P2
I
I
normal
apo A1
gene
apo A1 cONA probe
Ri
mutant
apo A1
gene
>-//
* Inserted DNA
Rs -1 L P 3
SIZE
(Kllobase Pairs)
GENE
ENZYME
Normal
Eco R1
R1-R2
Normal
Pst 1
P1-P2
Mutant
Eco R1
R3-R2
6.5
Ps-P2
0.95
Mutant
Pst 1
Figure 7.
FRAGMENT
13
2.2
Structure of the normal and mutant apo A-l gene. See text for details.
224
ARTERIOSCLEROSIS
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probe that were used for the blot in Figure 6. The
mutant apo A-l gene is now known to contain a DNA
insertion of 7.5 kbp within exon IV that disrupts production of the apo A-l protein. 35 '" Whereas the normal gene yields an Eco Rl fragment of 13 kbp (fragment R1-R2), the mutant gene yields a fragment of
6.5 kbp (fragment R3-R2) due to the presence of an
Eco Rl site in the inserted DNA. Note that the R1-R3
fragment was not detected (Figure 6, Lanes F and G)
because the cDNA probe lies completely to one side
of the inserted DNA (Figure 7). The normal and mutant Pst I fragments that were detected with the
cDNA probe are also shown (Figure 7).
In this example, the altered phenotype is clearly
due to the DNA rearrangement detected as a rare
RFLP. This is an excellent example of how molecular
biology has been used to link a specific genetic lesion with premature coronary artery disease.7677
Further studies showed that the DNA inserted in the
apo A-l gene was derived from the apo C-lll gene
which lies adjacent to the apo A-l gene.35
A common RFLP has been identified in the human
apo C-ll gene after digestion with the enzyme Taq
I.78'79 This RFLP was used to show linkage between
the apo C-ll and apo E genes78'79 and to demonstrate
linkage between the apo C-ll gene and apo C-ll deficiency.80 Rees et al.81 also have detected a polymorphism in the flanking region of the apo A-l gene that
was subsequently localized to a single base change
in the 3' noncoding region of the adjacent apo C-lll
gene.24 This polymorphism was present at higher
frequency in hypertriglyceridemic patients although
additional studies are required to confirm this finding.
These initial studies of RFLP illustrate the potential
of such analysis with genes coding for apolipoproteins and enzymes of lipid metabolism. Large patient
populations characterized for disorders of lipid and
lipoprotein metabolism are available through various
lipid clinics in the United States. The analysis of DNA
from these patients will probably yield new information relating specific changes in the DNA to phenotypic changes in lipid metabolism. In some cases this
approach may lead to the molecular basis for the
defect. In other cases RFLP are likely to provide
further subcategorization of lipid disorders on the
basis of DNA alterations. Such information may be
useful in diagnosis and will provide leads for further
research.
RFLP has proven useful in the diagnosis of sickle
cell anemia because a specific restriction site polymorphism occurs in the 3' flanking region of the human beta globin gene.82 Prenatal diagnosis of beta
thalassaemias also takes advantage of polymorphisms in the globin gene locus.83 Recent studies
indicate that RFLP may be used for prenatal detection of an altered phenylalanine hydroxylase gene
associated with phenylketonuria.84 In addition to the
use of polymorphisms occurring within a restriction
enzyme recognition sequence, knowledge of the nucleotide sequence may permit researchers to design
specific DNA probes to detect an altered gene. For
5, No 3,
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1985
example, Conner et al.85 have designed specific oligonucleotide probes that can distinguish the sickle
cell beta S allele from the normal beta A allele. One
synthetic 19 nucleotide probe formed a perfect hybrid with the sickle cell DNA but did not hybridize at
all to the beta A DNA due to a single base mismatch.
Thus, it is possible to design probes for the detection
of point mutations. One could also design probes to
screen for specific insertions or deletions in the DNA.
While many of these procedures are now performed
in the research laboratory, it is not difficult to envision
the extension of such procedures to the clinical testing situation. This potential for arteriosclerosis research should develop rapidly as relevant genes are
characterized and as RFLP are identified and related
to specific metabolic disorders.
Reverse Genetics
Reverse genetics refers to the process through
which a specific DNA sequence is altered in vitro and
subsequently expressed in vivo to evaluate the functional signifiance of the alteration. The evaluation of
function might be at the nucleic acid level or at the
level of the protein product depending upon the particular question being asked. The ability to express a
gene that has been mutated at specific sites enables
the investigtor to approach structure-function relationships with a level of discrimination not previously
possible. The typical approach in such studies is to
carry out site-directed mutagenesis on an isolated
gene to produce a point mutation, deletion, or insertion in the DNA. The altered DNA is constructed into
an expression vector and introduced into an appropriate host cell for expression. The evaluation of the
engineered mutation might be performed by monitoring function within the host cell or might require purification of the mutated gene product. Reverse genetics has been used primarily to identify the DNA
sequences necessary for transcription, the processing of primary mRNA transcripts, and the regulation
of gene expression. However, recent studies have
shown the potential of reverse genetics for evaluating structure-function relationships at the protein
level.
This approach has been used by Villafranca et
al.86 to examine the role of specific amino acid residues in the catalytic activity of dihydrofolate reductase (DHFR) from Escherichia coli. Site-directed mutagenesis was carried out to systematically change
three specific amino acid residues within the 159
residue DHFR polypeptide. The specific residues
mutated were selected on the basis of the X-ray crystal structure of DHFR to provide information about
the mechanism of catalysis and about the folding of
the polypeptide chain. After in vitro mutagenesis, the
altered DNA was constructed into a plasmid vector
and expressed to high levels in E. coli. Subsequently, DHFR was purified from the E. coli and evaluated
for activity. With an efficient plasmid expression vector, up to 8 mg of purified DHFR was obtained from a
MOLECULAR BIOLOGY AND ARTERIOSCLEROSIS
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1 liter culture, thereby providing sufficient enzyme for
studies of catalytic activity and physical properties.
These initial experiments clearly illustrate the potential of this strategy for structure-function studies at
the protein level. In the example cited above, the
crystal structure of DHFR and a wealth of information
about the mechanism of catalysis provided the
groundwork for designing mutations and asking very
directed questions about the enzyme.
Application of a similar strategy to a far less defined situation has also been highly informative in
evaluating the biochemical activities of the human
transforming protein, ras p21. In these studies, constructs were made to express the normal cellular ras
protein or the oncogenic variant which has a valine
substituted for glycine at residue 12 of the polypeptide.87 Expression of these constructs in E. coli permitted the investigators to compare the biochemical
properties of the normal and oncogenic ras proteins
in crude extracts of the bacteria. These comparisons
showed no differences in the binding of GTP or the
autophosphorylation activity of the ras proteins.
However, the experiments revealed a previously unknown GTPase activity in the normal ras p21 that
was significantly reduced in the oncogenic ras p21, 87
For the proteins listed in Table 1, similar approaches could be used to define amino acid residues or sequence domains required for fundamental
activities such as apolipoprotein interactions with lipoproteins, enzyme substrate interactions, or the association of ligands such as apo E or growth factors
with their receptors. In the case of apo E, analyses of
variant proteins and chemically modified proteins
have identified a polypeptide domain involved in
binding to the apo B/E and the hepatic apo E receptor.45'464988 Site-directed mutagenesis should prove
useful in extending the analysis of this domain as
well as in evaluating other regions of the apo E
polypeptide.
Another application of reverse genetics at the protein level is in studies of intracellular protein metabolism. By expressing a mutant protein in a mammalian
host cell, one could address questions relating to
protein biosynthesis, intracellular protein topogenesis, protein turnover, or the assembly of macromolecular complexes. For example, apo A-1 is synthesized with a 6 amino acid terminal prosegment that is
not cleaved until after the protein is secreted.89"91
Expression experiments with mutant apo A-1 constructs in liver cell cultures could be carried out to
determine whether the prosegment plays a role in
the intracellular transport or secretion of apo A-1.
Similarly, expression experiments could be used
to examine the polypeptide regions responsible for
the plasma membrane localization of lipoprotein receptors or the endoplasmic reticulum localization of
HMG CoA reductase. Previously, problems of this
nature could only be approached by correlating biochemical changes with amino acid sequence alterations in naturally occurring variants. The development of procedures for site-directed mutagenesis
Williams
225
and in vivo expression systems now permits researchers to ask directly how specific mutations influence biochemical function. This is a powerful experimental tool that will permit investigators to
examine structure-function relationships at the protein level in a systematic way.
References
1. Manlatls T, Frltsch EF, Sambrook J. Molecular cloning. A
laboratory manual. Cold Spring Harbor Laboratory, 1982:
1-545
2. Old RW, Primrose SB. Principles of gene manipulation.
Berkeley: University of California Press, 1981:1-250
3. Bolivar F, Backman K. Plasmids of Eschenchia coli as cloning vectors. Methods Enzymol 1979;68:245-267
4. Wlerlnga B, Roskam W, Arnberg A, van der Zwaag-Gerrltsen J, Ab G, Gruber M. Purification of the mRNA for chicken
very low density lipoprotein II and molecular cloning of its fulllength double stranded cDNA. Nucleic Acids Res 1979;
7:2147-2163
5. Chan L, Dugalczyk A, Means AR. Molecular cloning of the
gene sequences of a major apoprotein in avian very low density lipoprotein. Biochemistry 1980;19:5631-5637
6. Wlskocll R, Bensky P, Dower W, Goldberger RF, Gordon
Jl, Deeley RG. Coordinate regulation of two estrogen-dependent genes in avian liver. Proc Natl Acad Sci USA 1980;
77:4474-^478
7. Protter AA, Wang S-Y, Shelness GS, Ostapchuk P, Williams DL. Isolation and characterization of a cDNA clone
specific for avian vitellogenin II. Nucleic Acids Res 1982;
10:4935-4950
8. Shoulders CC, Baralle FE. Isolation of the human HDL apeprotein Al gene. Nucleic Acids Res 1982;10:4873—^882
9. Breslow JL, Ross D, McPherson J, et al: Isolation and
characterization of cDNA clones for human apolipoprotein AI. Proc Natl Acad Sci USA 1982;79:6861-6865
10. Cheung P, Chan L. Nucleotide sequence of cloned cDNA of
human apolipoprotein A-l. Nucleic Acids Res 1983;11:37033715
11. Law SW, Gray G, Brewer HB Jr. cDNA cloning of human
apo A-1: amino add sequence of preproapo Al. Biochem
Biophys Res Commun 1983;112:257-264
12. Miller JCE, Barth RK, Shaw PH, Elliott R, Hastle ND. Identification of a cDNA clone for mouse apoprotein A-l (apoA-l)
and its use in characterization of apo Al mRNA expression in
liver and small intestine. Proc Natl Acad Sci USA 1983;
80:1511-1515
13. Breslow JL, McPherson J, Karathanasls SK, Zannls VI.
Identification and DNA sequence of a human apolipoprotein
E cDNA clone. J Biol Chem 1982257:14639-14641
14. Wallls SC, Rogne S, Gill L, et al. The isolation of cDNA
clones for human apolipoprotein E and the detection of apo E
RNA in hepatic and extra-hepatic tissues. EMBO J 1983;
:2369-2373
McLean JW, Elshourbagy NA, Chang DJ, Mahley RW,
Taylor JM. Human apolipoprotein E mRNA. cDNA cloning
and nucleotide sequencing of a new varient. J Biol Chem
1984;259:6498-6504
16. McLean JW, Fukazawa C, Taylor JM. Rat apolipoprotein E
mRNA. Cloning and sequencing of double-stranded cDNA. J
Biol Chem 1983;258:8993-9000
17. de Jong FA, Howlett G, Aldred AR, Fldge N, Schrelber G.
Synthesis of rat apolipoprotein E by Eschenchia coli infected
with recombinant bacteriophage. Biochem Biophys Res
Commun 1984;119:657-662
18. Reue KL, Quon DH, O'Donnell KA, Dlzlkes GJ, Fa reed
GC, Lusls AJ. Cloning and regulation of messenger RNA for
mouse apolipoprotein E. J Biol Chem 1984;259:2100-2107
226
ARTERIOSCLEROSIS
VOL
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
19. Gordon Jl, Smith DP, Alpers DH, Strauss AW. Cloning of a
complementary deoxyribonucleic acid encoding a portion of
rat intestinal preapolipoprotein AIV messenger ribonucleic
acid. Biochemistry 1982;21:5424-5431
20. Sharpe CR, Sldoll A, Shelley CS, Lucero MA, Shoulders
CC, Baralle FE. Human apolipoproteins Al, All, CM and CHI.
cDNA sequences and mRNA abundance. Nucleic Adds Res
1984;12:3917-3932
21. Jackson CL, Bruns GAP, Breslow JL. Isolation and sequence of a human apolipoprotein CM cDNA clone and its use
to isolate and map to human chromosome 19 the gene for
apolipoprotein CM. Proc Natl Acad Sci USA 1984;81:29452949
22. Myklebost O, Williamson B, Markham AF, et al. The isolation and characterization of cDNA clones for human apolipoprotein Cll. J Biol Chem 1984;259:4401-4404
23. Knott TJ, Robertson ME, Priestley LM, Urdea M, Wallls S,
Scott J. Characterization of mRNAs encoding the precursor
for human apolipoprotein Cl. Nucleic Acids Res 1984;12:
3909-3915
24. Karathanasls SK, McPherson J, Zannls VI, Breslow JL.
Linkage of human apolipoproteins Al and C-lll genes. Nature
1983:304:371-373
25. Chin DJ, Luskey KL, Faust JR, MacDonald RJ, Brown MS,
Goldstein JL. Molecular cloning of 3-hydroxy-3-methylglutaryl coenzyme A reductase and evidence for regulation of its
mRNA. Proc Natl Acad Sci USA 1982:79:7704-7708
26. Russell DW, Yamamoto T, Schneider WJ, Slaughter CJ,
Brown MS, Goldstein JL. cDNA cloning of the bovine low
density lipoprotein receptor: feedback regulation of a receptor
mRNA. Proc Natl Acad Sci USA 1983:80:7501-7505
27. Yamamoto T, Davis CG, Brown MS, et al. The human LDL
receptor: A cysteine-rich protein with multiple ALU sequences in its mRNA. Cell 1984:39:27-38
28. Young RA, Davis RW. Efficient isolation of genes by using
antibody probes. Proc Natl Acad Sci USA 1983:8011941198
29 Helfman DM, Feramlsco JR, Flddes JC, Thomas GP,
Hughes SH. Identification of clones that encode chicken tropomyosin by direct immunological screening of a cDNA expression library. Proc Natl Acad Sci USA 1983:80.31-35
30. Manlatis T, Hardlson RC, Lacy C, et al. The isolation of
structural genes from libraries of eucaryotic DNA. Cell 1978;
15 687-701
31. Meljllnk FCPW, van het Schip AD, Arnberg AC, Wlerlnga
B, Ab G, Gruber M. Structure of the chicken apo very low
density lipoprotein II gene. J Biol Chem 1981,256:9668-9671
32. Wiskocil R, Goldman P, Deeley RG. Cloning and structural
characterization of an estrogen-dependent apolipoprotein
gene. J Biol Chem 1981 ;256:9662-9667
33. Karathanasis SK, Zannis VI, Breslow JL. Isolation and
characterization of the human apolipoprotein A-l gene. Proc
Natl Acad Sci USA 1983:80:6147-6151
34. Shoulders CC, Kornbllhtt AR, Munro BS, Baralle FE.
Gene structure of human apolipoprotein Al. Nucleic Acids
Res 1983:11:2827-2837
35. Breslow JL. Human apolipoprotein molecular biology and
genetic variation. Ann Rev Biochem 1985,54:699-727
36. Reynolds GA, Basu SK, Osborne TF, et al. HMG CoA
reductase: a negatively regulated gene with unusual promotor and 5' untranslated regions. Cell 1984:38:275-285
37. van het Schlp AD, Mei|llnk FCPW, Strijker R, et al. The
nucleotide sequence of the chicken apo very low density
lipoprotein II gene. Nucleic Acids Res 1983:11:2529-2540
38. Kane JP. Apolipoprotein B: structural and metabolic heterogeneity. Ann Rev Physiol 1983:45:637-650
39. LeBoeuf RC, Miller C, Shively JE, Schumaker VN, Balla
MA, Lusis AJ. Human apolipoprotein B: partial amino acid
sequence. FEBS Lett 1984;170:105-108
40. Brown MS, Goldstein JL. Multivalent feedback regulation of
HMG CoA reductase, a control mechanism coordinating isoprenoid synthesis and cell growth. J LJpid Res 1980;21:
505-517
41. Chin DJ, Gil G, Russell DW, et al. Nucleotide sequence of 3hydroxy-3 methyl-glutaryl coenzyme A reductase, a glycopro-
5, No 3,
MAY/JUNE
1985
tein of endoplasmic reticulum. Nature 1984;308:613-617
43. Llscum L, Finer-Moore J, Stroud RM, Luskey KL, Brown
MS, Goldstein JL. Domain structure of 3-hydroxy-3-methylglutaryl coenzyme A reductase, a glycoprotein of the endoplasmic reticulum. J Biol Chem 1985:260:522-530
43 Utermann G, Vogelberg KH, Stelnmetz A, et al. Polymorphism of apolipoprotein E. Genetics of hyperiipoproteinemia
type III. Clin Genet 1979;15:37-62
44. Zannls VI, Breslow JL. Human very low density lipoprotein
apolipoprotein E isoprotein polymorphism is explained by genetic variation and post-translational modification. Biochemistry 1981:20:1033-1041
45. Welsgraber KH, Rail SC Jr, Mahley RW. Human E apoprotein heterogeneity. Cysteine-arginine interchanges in the
ammo acid sequence of the apo-E isoforms. J Biol Chem
1981:256:9077-9083
46. Rail SC Jr, Weisgraber KH, Innerarlty TL, Mahley RW.
Structural basis for receptor binding heterogeneity of apolipoprotein E from type III hyperiipoproteinemic subjects. Proc
Natl Acad Sci USA 1982;79:4696-4700
47. Rail SC Jr, Welsgraber KH, Mahley RW. Human apolipoprotein E. The complete ammo acid sequence. J Biol Chem
1982:257:4171-4178
48. Das HK, McPherson J, Bruns GAP, Karathanasls SK,
Breslow JL. Isolation, characterization, and mapping to
chromosome 19 of the human apolipoprotein E gene. J Biol
Chem 1985 (in press)
49 Hul DY, Innerarlty TL, Mahley RW. Defective hepatic lipoprotein receptor binding of beta-very low density lipoproteins
from Type III hyperiipoproteinemic patients. Importance of
apolipoprotein E. J Biol Chem 1984:259:860-869
50 Utermann G, Jaeschke M, Menzel J. Familial hyperiipoproteinemia Type III: deficiency of a specific apolipoprotein (apo
E-lll) in the very low density lipoproteins. FEBS Lett 1975;
56.352-355
51. Pagnan A, Havel RJ, Kane JP, Kotlte L. Characterization of
human very low density lipoproteins containing two electrcphoretic populations: double prebeta lipoproteinemia and primary dysbetalipoproteinemia. J LJpid Res 1977;18:613-622
52 Zannls VI, Breslow JL. Characterization of a unique human
apolipoprotein E variant associated with type III hyperiipoproteinemia J Biol Chem 1980:255:1759-1762
53. Niman HL, Houghten RA, Walker LE, et al. Generation of
protein reactive antibodies by short peptides is an event of
high frequency: Implications for the structural basis of immune recognition Proc Natl Acad Sci USA 1983;80:49494953
54 Cohen GH, Dietzschold B, Ponce de Leon M, et al. Localization and synthesis of an antigenic determinant of Herpes
simplex virus glycoprotein D that stimulates the production of
neutralizing antibody. J Virol 1984;49-102-108
55. Emlni EA, Jameson BA, Wlmmer E. Priming for and induction of antipoliovirus neutralizing antibodies by synthetic peptides. Nature 1983:304:699-703
56 Hopp TP, Woods KR. Prediction of protein antigenic determinants from ammo acid sequences. Proc Natl Acad Sci USA
1981;78:3824-3828
57. Russell DW, Schneider WJ, Yamamoto T, Luskey KL,
Brown MS, Goldstein JL. Domain map of the LDL receptorsequence homology with the epidermal growth factor precursor. Cell 1984:37:577-585
58. Chan L, Jackson RL, O'Malley BW, Means AR. Synthesis
of very low density lipoproteins in the cockerel. Effects of
estrogen. J Clin Invest 1976:58:368-379
59. Chan L, Bradley WA, Means AR. Amino acid sequence of
the signal peptide of apo VLDL-II, a major apoprotem in avian
very low density lipoproteins. J Biol Chem 1980;255:
10060-10063
60. Dugaiczyk A, Inglis AS, Strike PM, Burley RW, Beattle
WG, Chan L. Comparison of the nucleotide sequence of
cloned DNA coding for an apolipoprotein (apo VLDL-II) from
avian blood and the amino acid sequence of an egg-yolk
protein (apovitellenin I): equivalence of the two sequences.
Gene 1981 ;14:175-182
61. Shelne88 GS, Williams DL. Apolipoprotein II messenger
MOLECULAR BIOLOGY AND ARTERIOSCLEROSIS
62.
63.
64.
65.
66.
67.
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
68.
69.
70.
71.
72.
73.
74.
75.
76.
77.
RNA. Transcriptional and splicing heterogeneity yields six 5'untranslated leader sequences. J Biol Chem 1984;259:99299935
Jackson RL, Chan L, Snow LD, Means AR. Hormonal regulation of lipoprotein synthesis. In: Dietschy JM, Gotto AM Jr,
Ontko JA. Disturbances in lipid and lipoprotein metabolism.
Bethesda: American Physiological Society, 1978; 139-154
Elbrecht A, Lazier CB, Protter AA, Williams DL. Independent developmental programs for two estrogen-regulated
genes. Science 1984;225:639-641
McKnight SL, Kingsbury R. Transciptional control signals of
a eucaryotic protein-coding gene. Science 1982;217:316324
Ross R. The arterial wall and atherosclerosis. Annu Rev Med
1979;30:1-15
Doolittle RF, Hunkapiller MW, Hood LE, et al. Simian sarcoma virus one gene, v-sis, is derived from the gene (or
genes) encoding a platelet-derived growth factor. Science
1983:221:275-277
Waterfield MD, Scrace GT, Whittle N, et al. Platelet-derived
growth factor is structurally related to the putative transforming protein p28sis of simian sarcoma virus. Nature 1983;
304:35-39
Assoian RK, Grotendorst GR, Miller DM, Sporn MB. Cellular transformation by coordinated action of three peptide
growth factors from human platelets. Nature 1984;309:804806
Kelley K, Cochran BH, Stiles CD, Leder P. Cell-specific
regulation of the c-myc gene by lymphocyte mitogens and
platelet-derived growth factor. Cell 1983;35:603-610
Blue M-L, Williams DL, Zucker S, Khan SA, Blum CB.
Apolipoprotein E synthesis in human kidney, adrenal gland,
and liver. Proc Natl Acad Sci USA 1983;80:283-287
Basu SK, Brown MS, Ho YK, Havel RJ, Goldstein JL.
Mouse macrophages synthesize and secrete a protein resembling apolipoprotein E. Proc Natl Acad Sci USA 1981;
78:7545-7549
Newman TC, Dawson PA, Rudel LL, Williams DL. Quantitation of apolipoprotein E mRNA in the liver and peripheral
tissues of nonhuman primates. J Biol Chem 1985;260:
2452-2457
McAllister LB, Scheller RH, Kandel ER, Axel R. In situ
hybridization to study the origin and fate of identified neurons.
Science 1983;222:800-808
Jeffreys AJ. DNA sequence variants in the G y-, A y-, 8-, and
13-globin genes of man. Cell 1979;18:1-10
Southern EM. Detection of specific sequences among DNA
fragments separated by gel electrophoresis. J Mol Biol 1975;
98:503-517
Karathanasis SK, Norum RA, Zannis VI, Breslow JL. An
inherited polymorphism in the human apolipoprotein A-l gene
locus related to the development of atherosclerosis. Nature
1983;301:718-720
Karathanasis SK, Zannis VI, Breslow JL. A DNA insertion
Index Terms: arteriosclerosis
• atherosclerosis
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
• cDNA
Williams
227
in the apolipoprotein A-l gene of patients with premature
atherosclerosis. Nature 1983:305:823-825
Myklebost O, Rogne S, Olaisen B, Gedde-Dahl T, Prydz H.
The locus for apolipoprotein Cll is closely linked to the apolipoprotein E locus on chromosome 19 in man. Hum Genet
1984:67:309-312
Humphries SE, Berg K, Gill L, et al. The gene for apolipoprotein C-ll is closely linked to the gene for apolipoprotein E
on chromosome 19. Clin Genet 1984:26:389-396
Humphries SE, Williams L, Myklebost O, et al. Familial
apolipoprotein Cll deficiency: A preliminary analysis of the
gene defect in two independent families. Hum Genet 1984;
67:151-155
Rees A, Shoulders CC, Stocks J, Galton DJ, Baralle FE.
DNA polymorphism adjacent to human apoprotein A-l gene:
relation to hypertriglyceridemia. Lancet 1983; 1:444—446
Kan YW, Dozy AM. Polymorphism of DNA sequence adjacent to human beta-globin structural gene: relationship to
sickle mutation. Proc Natl Acad Sci USA 1978;75:5631-5635
Orkin SH, Kazazian HH Jr, Antonarakis SE, et al. Linkage
of beta-thalassaemia mutations and beta-globin gene polymorphisms with DNA polymorphisms in human beta-globin
gene cluster. Nature 1982;296:627-631
Woo SLC, Lidsky AS, Guttler F, Chandra T, Robson KJH.
Cloned human phenylalanine hydroxylase gene allows prenatal diagnosis and carrier detection of classical phenylketonuria. Nature 1983;306:151-155
Conner BJ, Reyes AA, Morin C, Itakura I, Teplitz RL, Wallace RB. Detection of sickle cell betas-globin allele by hybridization with synthetic oligonucleotides. Proc Natl Acad Sci
USA 1983;80:;278-282
Villafranca JE, Howell EE, Voet DH, et al. Directed mutagenesis of dihydrofolate reductase. Science 1983:222:782788
McGrath JP, Capon DJ, Goeddel DV, Levinson AD. Comparative biochemical properties of normal and activated human ras p21 protein. Nature 1984;310:644-649
Weisgraber KH, Innerarity TL, Mahley RW. Abnormal lipoprotein receptor-binding activity of the human E apoprotein
due to cysteine-arginine interchange at a single site. J Biol
Chem 1982;257:2518-2521
Gordon Jl, Smith DP, Andy R, Alpers DH, Schonfeld G,
Strauss AW. The primary translation product of rat intestinal
apolipoprotein Al mRNA is an unusual preproprotein. J Biol
Chem 1982;257:971-978
Gordon Jl, Sims HF, Lentz SR, Edelstein C, Scanu AM,
Strauss AW. Proteolytic processing of human preproapolipoprotein A-l. A proposed defect in the conversion of pro A-l to
A-l in Tangier's disease. J Biol Chem 1983;258:4037-4044
Zannis VI, Karathanasis SK, Kreutmann H, Goldberger G,
Breslow JL. Intracellular and extracellular processing of human apolipoprotein A-l: secreted apolipoprotein A-l isoprotein 2 is a propeptide. Proc Natl Acad Sci USA 1983;
80:2574-2578
• gene
• molecular biology
• apolipoprotein
Molecular biology in arteriosclerosis research.
D L Williams
Downloaded from http://atvb.ahajournals.org/ by guest on June 18, 2017
Arterioscler Thromb Vasc Biol. 1985;5:213-227
doi: 10.1161/01.ATV.5.3.213
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