Overexpression of mammalian and viral proteins

The invention features a synthetic gene encoding a protein normally expressed in mammalian cells wherein at least one non-preferred or less preferred codon in the natural gene encoding the mammalian protein has been replaced by a preferred codon encoding the same amino acid.

FIELD OF THE INVENTION 
The invention concerns genes and methods for expressing eukaryotic and 
viral proteins at high levels in eukaryotic cells. 
BACKGROUND OF THE INVENTION 
Expression of eukaryotic gene products in prokaryotes is sometimes limited 
by the presence of codons that are infrequently used in E. coli. 
Expression of such genes can be enhanced by systematic substitution of the 
endogenous codons with codons overrepresented in highly expressed 
prokaryotic genes (Robinson et al. 1984). It is commonly supposed that 
rare codons cause pausing of the ribosome, which leads to a failure to 
complete the nascent polypeptide chain and an uncoupling of transcription 
and translation. The mRNA 3' end of the stalled ribosome is exposed to 
cellular ribonucleases, which decreases the stability of the transcript. 
SUMMARY OF THE INVENTION 
The invention features a synthetic gene encoding a protein normally 
expressed in mammalian cells wherein at least one non-preferred or less 
preferred codon in the natural gene encoding the mammalian protein has 
been replaced by a preferred codon encoding the same amino acid. 
Preferred codons are: Ala (gcc); Arg (cgc); Asn (aac); Asp (gac) Cys (tgc); 
Gln (cag); Gly (ggc); His (cac); Ile (atc); Leu (ctg); Lys (aag); Pro 
(ccc); Phe (ttc); Ser (agc); Thr (acc); Tyr (tac); and Val (gtg). Less 
preferred codons are: Gly (ggg); Ile (att); Leu (ctc); Ser (tcc); Val 
(gtc). All codons which do not fit the description of preferred codons or 
less preferred codons are non-preferred codons. 
By "protein normally expressed in mammalian cells" is meant a protein which 
is expressed in mammalian cells under natural conditions. The term 
includes genes in the mammalian genome encoding polypeptides such as 
Factor VIII, Factor IX, interleukins, and other proteins. The term also 
includes genes which are expressed in a mammalian cell under disease 
conditions such as oncogenes as well as genes which are encoded by a virus 
(including a retrovirus) which are expressed in mammalian cells 
post-infection 
In preferred embodiments, the synthetic gene is capable of expressing said 
mammalian protein at a level which is at least 110%, 150%, 200%, 500%, 
1,000%, or 10,000% of that expressed by said natural gene in an in vitro 
mammalian cell culture system under identical conditions (i.e., same cell 
type, same culture conditions, same expression vector). 
Suitable cell culture systems for measuring expression of the synthetic 
gene and corresponding natural gene are described below. Other suitable 
expression systems employing mammalian cells are well known to those 
skilled in the art and are described in, for example, the standard 
molecular biology reference works noted below. Vectors suitable for 
expressing the synthetic and natural genes are described below and in the 
standard reference works described below. By "expression" is meant protein 
expression. Expression can be measured using an antibody specific for the 
protein of interest. Such antibodies and measurement techniques are well 
known to those skilled in the art. By "natural gene" is meant the gene 
sequence which naturally encodes the protein. 
In other preferred embodiments at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 
80%, or 90% of the codons in the natural gene are non-preferred codons. 
In a preferred embodiment the protein is a retroviral protein. In a more 
preferred embodiment the protein is a lentiviral protein. In an even more 
preferred embodiment the protein is an HIV protein. In other preferred 
embodiments the protein is gag, pol, env, gp120, or gp160. In other 
preferred embodiments the protein is a human protein. 
The invention also features a method for preparing a synthetic gene 
encoding a protein normally expressed by mammalian cells. The method 
includes identifying non-preferred and less-preferred codons in the 
natural gene encoding the protein and replacing one or more of the 
non-preferred and less-preferred codons with a preferred codon encoding 
the same amino acid as the replaced codon. 
Under some circumstances (e.g., to permit introduction of a restriction 
site) it may be desirable to replace a non-preferred codon with a less 
preferred codon rather than a preferred codon. 
It is not necessary to replace all less preferred or non-preferred codons 
with preferred codons. Increased expression can be accomplished even with 
partial replacement. 
In other preferred embodiments the invention features vectors (including 
expression vectors) comprising the synthetic gene. 
By "vector" is meant a DNA molecule, derived, e.g., from a plasmid, 
bacteriophage, or mammalian or insect virus, into which fragments of DNA 
may be inserted or cloned. A vector will contain one or more unique 
restriction sites and may be capable of autonomous replication in a 
defined host or vehicle organism such that the cloned sequence is 
reproducible. Thus, by "expression vector" is meant any autonomous element 
capable of directing the synthesis of a protein. Such DNA expression 
vectors include mammalian plasmids and viruses. 
The invention also features synthetic gene fragments which encode a desired 
portion of the protein. Such synthetic gene fragments are similar to the 
synthetic genes of the invention except that they encode only a portion of 
the protein. Such gene fragments preferably encode at least 50, 100, 150, 
or 500 contiguous amino acids of the protein. 
In constructing the synthetic genes of the invention it may be desirable to 
avoid CpG sequences as these sequences may cause gene silencing. 
The codon bias present in the HIV gp120 envelope gene is also present in 
the gag and pol proteins. Thus, replacement of a portion of the 
non-preferred and less preferred codons found in these genes with 
preferred codons should produce a gene capable of higher level expression. 
A large fraction of the codons in the human genes encoding Factor VIII and 
Factor IX are non-preferred codons or less preferred codons. Replacement 
of a portion of these codons with preferred codons should yield genes 
capable of higher level expression in mammalian cell culture. Conversely, 
it may be desirable to replace preferred codons in a naturally occurring 
gene with less-preferred codons as a means of lowering expression. 
Standard reference works describing the general principles of recombinant 
DNA technology include Watson, J. D. et al., Molecular Biology of the 
Gene, Volumes I and II, the Benjamin/Cummings Publishing Company, Inc., 
publisher, Menlo Park, Calif. (1987); Darnell, J. E. et al., Molecular 
Cell Biology, Scientific American Books, Inc., Publisher, New York, N.Y. 
(1986); Old, R. W., et al., Principles of Gene Manipulation: An 
Introduction to Genetic Engineering, 2d edition, University of California 
Press, publisher, Berkeley, Calif. (1981); Maniatis, T., et al., Molecular 
Cloning: A Laboratory Manual, 2nd Ed. Cold Spring Harbor Laboratory, 
publisher, Cold Spring Harbor, N.Y. (1989); and Current Protocols in 
Molecular Biology, Ausubel et al., Wiley Press, New York, N.Y. (1989).

DESCRIPTION OF THE PREFERRED EMBODIMENTS 
Construction of a Synthetic gp120 Gene Having Codons Found in Highly 
Expressed Human Genes 
A codon frequency table for the envelope precursor of the LAV subtype of 
HIV-1 was generated using software developed by the University of 
Wisconsin Genetics Computer Group. The results of that tabulation are 
contrasted in Table 1 with the pattern of codon usage by a collection of 
highly expressed human genes. For any amino acid encoded by degenerate 
codons, the most favored codon of the highly expressed genes is different 
from the most favored codon of the HIV envelope precursor. Moreover a 
simple rule describes the pattern of favored envelope codons wherever it 
applies: preferred codons maximize the number of adenine residues in the 
viral RNA. In all cases but one this means that the codon in which the 
third position is A is the most frequently used. In the special case of 
serine, three codons equally contribute one A residue to the mRNA; 
together these three comprise 85% of the codons actually used in envelope 
transcripts. A particularly striking example of the A bias is found in the 
codon choice for arginine, in which the AGA triplet comprises 88% of all 
codons. In addition to the preponderance of A residues, a marked 
preference is seen for uridine among degenerate codons whose third residue 
must be a pyrimidine. Finally, the inconsistencies among the less 
frequently used variants can be accounted for by the observation that the 
dinucleotide CpG is underrepresented; thus the third position is less 
likely to be G whenever the second position is C, as in the codons for 
alanine, proline, serine and threonine; and the CGX triplets for arginine 
are hardly used at all. 
TABLE 1 
______________________________________ 
Codon Frequency in the HIV-1 IIIb env gene and 
in highly expressed human genes. 
High Env 
______________________________________ 
Ala 
GC C 53 27 
T 17 18 
A 13 50 
G 17 5 
Arg 
CG C 37 0 
T 7 4 
A 6 0 
G 21 0 
AG A 10 88 
G 18 8 
Asn 
AA C 78 30 
T 22 70 
Asp 
GA C 75 33 
T 25 67 
Leu 
CT C 26 10 
T 5 7 
A 3 17 
G 58 17 
TT A 2 30 
G 6 20 
Lys 
AA A 18 68 
G 82 32 
Pro 
CC C 48 27 
T 19 14 
A 16 55 
G 17 5 
Phe 
TT C 80 26 
T 20 74 
Cys 
TG C 68 16 
T 32 84 
Gln 
CA A 12 55 
G 88 45 
Glu 
GA A 25 67 
G 75 33 
Gly 
GG C 50 6 
T 12 13 
A 14 53 
G 24 28 
His 
CA C 79 25 
T 21 75 
Ile 
AT C 77 25 
T 18 31 
A 5 44 
Ser 
TC C 28 8 
T 13 8 
A 5 22 
G 9 0 
AG C 34 22 
T 10 41 
Thr 
AC C 57 20 
T 14 22 
A 14 51 
G 15 7 
Tyr 
TA C 74 8 
T 26 92 
Val 
GT C 25 12 
T 7 9 
A 5 62 
G 64 18 
______________________________________ 
Codon frequency was calculated using the GCG program established the the 
University of Wisconsin Genetics Computer Group. Numbers represent the 
percentage of cases in which the particular codon is used. Codon usage 
frequencies of envelope genes of other HIV1 virus isolates are comparable 
and show a similar bias. 
In order to produce a gp120 gene capable of high level expression in 
mammalian cells, a synthetic gene encoding the gp120 segment of HIV-1 was 
constructed (syngp120mn), based on the sequence of the most common North 
American subtype, HIV-1 MN (Shaw et al. 1984; Gallo et al. 1986). In this 
synthetic gp120 gene nearly all of the native codons have been 
systematically replaced with codons most frequently used in highly 
expressed human genes (FIG. 1). This synthetic gene was assembled from 
chemically synthesized oligonucleotides of 150 to 200 bases in length. If 
oligonucleotides exceeding 120 to 150 bases are chemically synthesized, 
the percentage of full-length product can be low, and the vast excess of 
material consists of shorter oligonucleotides. Since these shorter 
fragments inhibit cloning and PCR procedures, it can be very difficult to 
use oligonucleotides exceeding a certain length. In order to use crude 
synthesis material without prior purification, single-stranded 
oligonucleotide pools were PCR amplified before cloning. PCR products were 
purified in agarose gels and used as templates in the next PCR step. Two 
adjacent fragments could be co-amplified because of overlapping sequences 
at the end of either fragment. These fragments, which were between 350 and 
400 bp in size, were subcloned into a pCDM7-derived plasmid containing the 
leader sequence of the CD5 surface molecule followed by a 
Nhe1/Pst1/Mlu1/EcoR1/BamH1 polylinker. Each of the restriction enzymes in 
this polylinker represents a site that is present at either the 5' or 3' 
end of the PCR-generated fragments. Thus, by sequential subcloning of each 
of the 4 long fragments, the whole gp120 gene was assembled. For each 
fragment 3 to 6 different clones were subcloned and sequenced prior to 
assembly. A schematic drawing of the method used to construct the 
synthetic gp120 is shown in FIG. 2. The sequence of the synthetic gp120 
gene (and a synthetic gp160 gene created using the same approach) is 
presented in FIG. 1. 
The mutation rate was considerable. The most commonly found mutations were 
short (1 nucleotide) and long (up to 30 nucleotides) deletions. In some 
cases it was necessary to exchange parts with either synthetic adapters or 
pieces from other subclones without mutation in that particular region. 
Some deviations from strict adherence to optimized codon usage were made 
to accommodate the introduction of restriction sites into the resulting 
gene to facilitate the replacement of various segments (FIG. 2). These 
unique restriction sites were introduced into the gene at approximately 
100 bp intervals. The native HIV leader sequence was exchanged with the 
highly efficient leader peptide of the human CD5 antigen to facilitate 
secretion. The plasmid used for construction is a derivative of the 
mammalian expression vector pCDM7 transcribing the inserted gene under the 
control of a strong human CMV immediate early promoter. 
To compare the wild-type and synthetic gp120 coding sequences, the 
synthetic gp120 coding sequence was inserted into a mammalian expression 
vector and tested in transient transfection assays. Several different 
native gp120 genes were used as controls to exclude variations in 
expression levels between different virus isolates and artifacts induced 
by distinct leader sequences. The gp120 HIV IIIb construct used as control 
was generated by PCR using a Sal1/Xho1 HIV-1 HXB2 envelope fragment as 
template. To exclude PCR induced mutations a Kpn1/Ear1 fragment containing 
approximately 1.2 kb of the gene was exchanged with the respective 
sequence from the proviral clone. The wildtype gp120mn constructs used as 
controls were cloned by PCR from HIV-1 MN infected C8166 cells (AIDS 
Repository, Rockville, Md.) and expressed gp120 either with a native 
envelope or a CD5 leader sequence. Since proviral clones were not 
available in this case, two clones of each construct were tested to avoid 
PCR artifacts. To determine the amount of secreted gp120 
semi-quantitatively supernatants of 293T cells transiently transfected by 
calcium phosphate coprecipitation were immunoprecipitated with soluble 
CD4:immunoglobulin fusion protein and protein A sepharose. 
The results of this analysis (FIG. 3) show that the synthetic gene product 
is expressed at a very high level compared to that of the native gp120 
controls. The molecular weight of the synthetic gp120 gene was comparable 
to control proteins (FIG. 3) and appeared to be in the range of 100 to 110 
kd. The slightly faster migration can be explained by the fact that in 
some tumor cell lines like 293T glycosylation is either not complete or 
altered to some extent. 
To compare expression more accurately gp120 protein levels were quantitated 
using a gp120 ELISA with CD4 in the demobilized phase. This analysis shows 
(FIG. 4) that ELISA data were comparable to the immunoprecipitation data, 
with a gp120 concentration of approximately 125 ng/ml for the synthetic 
gp120 gene, and less than the background cutoff (5 ng/ml) for all the 
native gp120 genes. Thus, expression of the synthetic gp120 gene appears 
to be at least one order of magnitude higher than wildtype gp120 genes. In 
the experiment shown the increase was at least 25 fold. 
The Role of rev in gp120 Expression 
Since rev appears to exert its effect at several steps in the expression of 
a viral transcript, the possible role of non-translational effects in the 
improved expression of the synthetic gp120 gene was tested. First, to rule 
out the possibility that negative signals elements conferring either 
increased mRNA degradation or nucleic retention were eliminated by 
changing the nucleotide sequence, cytoplasmic mRNA levels were tested. 
Cytoplasmic RNA was prepared by NP40 lysis of transiently transfected 293T 
cells and subsequent elimination of the nuclei by centrifugation. 
Cytoplasmic RNA was subsequently prepared from lysates by multiple phenol 
extractions and precipitation, spotted on nitrocellulose using a slot blot 
apparatus, and finally hybridized with an envelope-specific probe. 
Briefly, cytoplasmic mRNA 293 cells transfected with CDM7, gp120IIIB, or 
syngp120 was isolated 36 hours post transfection. Cytoplasmic RNA of Hela 
cells infected with wildtype vaccinia virus or recombinant virus 
expressing gp120IIIb or the synthetic gp120 gene was under the control of 
the 7.5 promoter was isolated 16 hours post infection. Equal amounts were 
spotted on nitrocellulose using a slot blot device and hybridized with 
randomly labelled 1.5 kb gp120IIIb and syngp120 fragments or human 
beta-actin. RNA expression levels were quantitated by scanning the 
hybridized membranes with a phosphoimager. The procedures used are 
described in greater detail below. 
This experiment demonstrated that there was no significant difference in 
the mRNA levels of cells transfected with either the native or synthetic 
gp120 gene. In fact, in some experiments cytoplasmic mRNA level of the 
synthetic gp120 gene was even lower than that of the native gp120 gene. 
These data were confirmed by measuring expression from recombinant vaccinia 
viruses. Human 293 cells or Hela cells were infected with vaccinia virus 
expressing wildtype gp120IIIb or syngp120mn at a multiplicity of infection 
of at least 10. Supernatants were harvested 24 hours post infection and 
immunoprecipitated with CD4:immunoglobin fusion protein and protein A 
sepharose. The procedures used in this experiment are described in greater 
detail below. 
This experiment showed that the increased expression of the synthetic gene 
was still observed when the endogenous gene product and the synthetic gene 
product were expressed from vaccinia virus recombinants under the control 
of the strong mixed early and late 7.5 k promoter. Because vaccinia virus 
mRNAs are transcribed and translated in the cytoplasm, increased 
expression of the synthetic envelope gene in this experiment cannot be 
attributed to improved export from the nucleus. This experiment was 
repeated in two additional human cell types, the kidney cancer cell line 
293 and HeLa cells. As with transfected 293T cells, mRNA levels were 
similar in 293 cells infected with either recombinant vaccinia virus. 
Codon Usage in Lentivirus 
Because it appears that codon usage has a significant impact on expression 
in mammalian cells, the codon frequency in the envelope genes of other 
retroviruses was examined. This study found no clear pattern of codon 
preference between retroviruses in general. However, if viruses from the 
lentivirus genus, to which HIV-1 belongs were analyzed separately, codon 
usage bias almost identical to that of HIV-1 was found. A codon frequency 
table from the envelope glycoproteins of a variety of (predominantly type 
C) retroviruses excluding the lentiviruses was prepared, and a codon 
frequency table created from the envelope sequences of four lentiviruses 
not closely related to HIV-1 (caprine arthritis encephalitis virus, equine 
infectious anemia virus, feline immunodeficiency virus, and visna virus) 
(Table 2). The codon usage pattern for lentiviruses is strikingly similar 
to that of HIV-1. In all cases but one, the preferred codon for HIV-1 is 
the same as the preferred codon for the other lentiviruses. The exception 
is proline, which is encoded by CCT in 41% of non-HIV lentiviral envelope 
residues, and by CCA in 40% of residues, a situation which clearly also 
reflects a significant preference for the triplet ending in A. The pattern 
of codon usage by the non-lentiviral envelope proteins does not show a 
similar predominance of A residues, and is also not as skewed toward third 
position C and G residues as is the codon usage for the highly expressed 
human genes. In general non-lentiviral retroviruses appear to exploit the 
different codons more equally, a pattern they share with less highly 
expressed human genes. 
TABLE 2 
______________________________________ 
Codon frequency in the envelope gene of 
lentiviruses (lenti) and non-lentiviral retroviruses (other). 
Other 
Lenti 
______________________________________ 
Ala 
GC C 45 13 
T 26 37 
A 20 46 
G 9 3 
Arg 
CG C 14 2 
T 6 3 
A 16 5 
G 17 3 
AG A 31 51 
G 15 26 
Asn 
AA C 49 31 
T 51 69 
Asp 
GA C 55 33 
T 51 69 
Leu 
CT C 22 8 
T 14 9 
A 21 16 
G 19 11 
TT A 15 41 
G 10 16 
Lys 
AA A 60 63 
G 40 37 
Pro 
CC C 42 14 
T 30 41 
A 20 40 
G 7 5 
Phe 
TT C 52 25 
T 48 75 
Cys 
TG C 53 21 
T 47 79 
Gln 
CA A 52 69 
C 48 31 
Glu 
GA A 57 68 
G 43 32 
Gly 
CC C 21 8 
T 13 9 
A 37 56 
G 29 26 
His 
CA C 51 38 
T 49 62 
Ile 
AT C 38 16 
T 31 22 
A 31 61 
Ser 
TC C 38 10 
T 17 16 
A 18 24 
G 6 5 
AG C 13 20 
T 7 25 
Thr 
AC C 44 18 
T 27 20 
A 19 55 
G 10 8 
Tyr 
TA C 48 28 
T 52 72 
Val 
GT C 36 9 
T 17 10 
A 22 54 
G 25 27 
______________________________________ 
Codon frequency was calculated using the GCG program established by the 
University of Wisconsin Genetics Computer Group. Numbers represent the 
percentage in which a particular codon is used. Codon usage of 
nonlentiviral retroviruses was compiled from the envelope precursor 
sequences of bovine leukemia virus feline leukemia virus, human Tcell 
leukemia virus type I, human Tcell lymphotropic virus type II, the mink 
cell focusforming isolate of murine leukemia virus (MuLV), the Rauscher 
spleen focusforming isolate, the 10A1 isolate, the 4070A amphotropic 
isolate and the myeloproliferative leukemia virus isolate, and from rat 
leukemia virus, simian sarcoma virus, simian Tcell leukemia virus, 
leukemogenic retrovirus T1223/B and gibbon ape leukemia virus. The codon 
frequency tables for the nonHIV, nonSIV lentiviruses were compiled from 
the envelope precursor sequences for caprine arthritis encephalitis virus 
equine infectious anemia virus, feline immunodeficiency virus, and visna 
virus. 
In addition to the prevalence of A containing codons, lentiviral codons 
adhere to the HIV pattern of strong CpG underrepresentation, so that the 
third position for alanine, proline, serine and threonine triplets is 
rarely G. The retroviral envelope triplets show a similar, but less 
pronounced, underrepresentation of CpG. The most obvious difference 
between lentiviruses and other retroviruses with respect to CpG prevalence 
lies in the usage of the CGX variant of arginine triplets, which is 
reasonably frequently represented among the retroviral envelope coding 
sequences, but is almost never present among the comparable lentivirus 
sequences. 
Differences in rev Dependence Between Native and Synthetic gp120 
To examine whether regulation by rev is connected to HIV-1 codon usage, the 
influence of rev on the expression of both native and synthetic gene was 
investigated. Since regulation by rev requires the rev-binding site RRE in 
cis, constructs were made in which this binding site was cloned into the 
3' untranslated region of both the native and the synthetic gene. These 
plasmids were co-transfected with rev or a control plasmid in trans into 
293T cells, and gp120 expression levels in supernatants were measured 
semiquantitatively by immunoprecipitation. The procedures used in this 
experiment are described in greater detail below. 
As shown in FIG. 5, panels A and B, rev upregulates the native gp120 gene, 
but has no effect on the expression of the synthetic gp120 gene. Thus, the 
action of rev is not apparent on a substrate which lacks the coding 
sequence of endogenous viral envelope sequences. 
Expression of a synthetic rat THY-1 gene with HIV envelope codons 
The above-described experiment suggest that in fact "envelope sequences" 
have to be present for rev regulation. In order to test this hypothesis, a 
synthetic version of the gene encoding the small, typically highly 
expressed cell surface protein, rat THY-1 antigen, was prepared. The 
synthetic version of the rat THY-1 gene was designed to have a codon usage 
like that of HIV gp120. In designing this synthetic gene AUUUA sequences, 
which are associated with mRNA instability, were avoided. In addition, two 
restriction sites were introduced to simplify manipulation of the 
resulting gene (FIG. 6). This synthetic gene with the HIV envelope codon 
usage (rTHY-1env) was generated using three 150 to 170 mer 
oligonucleotides (FIG. 7). In contrast to the syngp120mn gene, PCR 
products were directly cloned and assembled in pUC12, and subsequently 
cloned into pCDM7. 
Expression levels of native rTHY-1 and rTHY-1 with the HIV envelope codons 
were quantitated by immunofluorescence of transiently transfected 293T 
cells. FIG. 8 shows that the expression of the native THY-1 gene is almost 
two orders of magnitude above the background level of the control 
transfected cells (pCDM7). In contrast, expression of the synthetic rat 
THY-1 is substantially lower than that of the native gene (shown by the 
shift of the peak towards a lower channel number). 
To prove that no negative sequence elements promoting mRNA degradation were 
inadvertently introduced, a construct was generated in which the rTHY-1env 
gene was cloned at the 3' end of the synthetic gp120 gene (FIG. 9, panel 
B). In this experiment 293T cells were transfected with either the 
syngp120mn gene or the syngp120/rat THY-1 env fusion gene 
(syngp120mn.rTHY-1env). Expression was measured by immunoprecipitation 
with CD4:IgG fusion protein and protein A agarose. The procedures used in 
this experiment are described in greater detail below. 
Since the synthetic gp120 gene has a UAG stop codon, rTHY-1env is not 
translated from this transcript. If negative elements conferring enhanced 
degradation were present in the sequence, gp120 protein levels expressed 
from this construct should be decreased in comparison to the syngp120mn 
construct without rTHY-1env. FIG. 9, panel A, shows that the expression of 
both constructs is similar, indicating that the low expression must be 
linked to translation. 
Rev-dependent expression of synthetic rat THY-1 gene with envelope codons 
To explore whether rev is able to regulate expression of a rat THY-1 gene 
having env codons, a construct was made with a rev-binding site in the 3' 
end of the rTHY1env open reading frame. To measure rev-responsiveness of 
the a rat THY-1env construct having a 3' RRE, human 293T cells were 
cotransfected with ratTHY-1envrre and either CDM7 or pCMVrev. At 60 hours 
post transfection cells were detached with 1 mM EDTA in PBS and stained 
with the OX-7 anti rTHY-1 mouse monoclonal antibody and a secondary 
FITC-conjugated antibody. Fluorescence intensity was measured using an 
EPICS XL cytofluorometer. These procedures are described in greater detail 
below. 
In repeated experiments, a slight increase of rTHY-1env expression was 
detected if rev was cotransfected with the rTHY-1env gene. To further 
increase the sensitivity of the assay system a construct expressing a 
secreted version of rTHY-1env was generated. This construct should produce 
more reliable data because the accumulated amount of secreted protein in 
the supernatant reflects the result of protein production over an extended 
period, in contrast to surface expressed protein, which appears to more 
closely reflect the current production rate. A gene capable of expressing 
a secreted form was prepared by PCR using forward and reverse primers 
annealing 3' of the endogenous leader sequence and 5' of the sequence 
motif required for phosphatidylinositol glycan anchorage respectively. The 
PCR product was cloned into a plasmid which already contained a CD5 leader 
sequence, thus generating a construct in which the membrane anchor has 
been deleted and the leader sequence exchanged by a heterologous (and 
probably more efficient) leader peptide. 
The rev-responsiveness of the secreted form ratTHY-1env was measured by 
immunoprecipitation of supernatants of human 293T cells cotransfected with 
a plasmid expressing a secreted form of ratTHY-1env and the RRE sequence 
in cis (rTHY-1envPI-rre) and either CDM7 or pCMVrev. The rTHY-1envPI-RRE 
construct was made by PCR using the oligonucleotides 
cgcggggctagcgcaaagagtaataagtttaac as forward and 
cgcggatcccttgtattttgtactaata a as reverse primers and the synthetic 
rTHY-1env construct as template. After digestion with Nhe1 and Not1 the 
PCR fragment was cloned into a plasmid containing CD5 leader and RRE 
sequences. Supernatants of .sup.35 S labelled cells were harvested 72 
hours post transfection, precipitated with a mouse monoclonal antibody OX7 
against rTHY-1 and anti-mouse IgG sepharose, and run on a 12% reducing 
SDS-PAGE. 
In this experiment the induction of rTHY-1env by rev was much more 
prominent and clearcut than in the above-described experiment and strongly 
suggests that rev is able to translationally regulate transcripts that are 
suppressed by low-usage codons. 
Rev-independent expression of a rTHY-1env:immunoglobulin fusion protein 
To test whether low-usage codons must be present throughout the whole 
coding sequence or whether a short region is sufficient to confer 
rev-responsiveness, a rTHY-1env:immunoglobulin fusion protein was 
generated. In this construct the rTHY-1env gene (without the sequence 
motif responsible for phosphatidylinositol glycan anchorage) is linked to 
the human IgG1 hinge, CH2 and CH3 domains. This construct was generated by 
anchor PCR using primers with Nhe1 and BamHI restriction sites and 
rTHY-1env as template. The PCR fragment was cloned into a plasmid 
containing the leader sequence of the CD5 surface molecule and the hinge, 
CH2 and CH3 parts of human IgG1 immunoglobulin. A Hind3/Eag1 fragment 
containing the rTHY-1enveg1 insert was subsequently cloned into a 
pCDM7-derived plasmid with the RRE sequence. 
To measure the response of the rTHY-1env/immunoglobin fusion gene 
(rTHY-1enveg1rre) to rev human 293T cells cotransfected with 
rTHY-1enveg1rre and either pCDM7 or pCMVrev. The rTHY-1enveg1rre construct 
was made by anchor PCR using forward and reverse primers with Nhe1 and 
BamH1 restriction sites respectively. The PCR fragment was cloned into a 
plasmid containing a CD5 leader and human IgG1 hinge, CH2 and CH3 domains. 
Supernatants of .sup.35 S labelled cells were harvested 72 hours post 
transfection, precipitated with a mouse monoclonal antibody OX7 against 
rTHY-1 and anti mouse IgG sepharose, and run on a 12% reducing SDS-PAGE. 
The procedures used are described in greater detail below. 
As with the product of the rTHY-1envPI-gene, this rTHY-1env/immunoglobulin 
fusion protein is secreted into the supernatant. Thus, this gene should be 
responsive to rev-induction. However, in contrast to rTHY-1envPI-, 
cotransfection of rev in trans induced no or only a negligible increase of 
rTHY-1enveg1 expression. 
The expression of rTHY-1:immunoglobulin fusion protein with native rTHY-1 
or HIV envelope codons was measured by immunoprecipitation. Briefly, human 
293T cells transfected with either rTHY-1enveg1 (env codons) or 
rTHY-1wteg1 (native codons). The rTHY-1wteg1 construct was generated in 
manner similar to that used for the rTHY-1enveg1 construct, with the 
exception that a plasmid containing the native rTHY-1 gene was used as 
template. Supernatants of .sup.35 S labelled cells were harvested 72 hours 
post transfection, precipitated with a mouse monoclonal antibody OX7 
against rTHY-1 and anti mouse IgG sepharose, and run on a 12% reducing 
SDS-PAGE. The procedures used in this experiment are described in greater 
detail below. 
Expression levels of rTHY-1enveg1 were decreased in comparison to a similar 
construct with wildtype rTHY-1 as the fusion partner, but were still 
considerably higher than rTHY-1env. Accordingly, both parts of the fusion 
protein influenced expression levels. The addition of rTHY-1env did not 
restrict expression to an equal level as seen for rTHY-1env alone. Thus, 
regulation by rev appears to be ineffective if protein expression is not 
almost completely suppressed. 
Codon preference in HIV-1 envelope genes 
Direct comparison between codon usage frequency of HIV envelope and highly 
expressed human genes reveals a striking difference for all twenty amino 
acids. One simple measure of the statistical significance of this codon 
preference is the finding that among the nine amino acids with two fold 
codon degeneracy, the favored third residue is A or U in all nine. The 
probability that all nine of two equiprobable choices will be the same is 
approximately 0.004, and hence by any conventional measure the third 
residue choice cannot be considered random. Further evidence of a skewed 
codon preference is found among the more degenerate codons, where a strong 
selection for triplets bearing adenine can be seen. This contrasts with 
the pattern for highly expressed genes, which favor codons bearing C, or 
less commonly G, in the third position of codons with three or more fold 
degeneracy. 
The systematic exchange of native codons with codons of highly expressed 
human genes dramatically increased expression of gp120. A quantitative 
analysis by ELISA showed that expression of the synthetic gene was at 
least 25 fold higher in comparison to native gp120 after transient 
transfection into human 293 cells. The concentration levels in the ELISA 
experiment shown were rather low. Since an ELISA was used for 
quantification which is based on gp120 binding to CD4, only native, 
non-denatured material was detected. This may explain the apparent low 
expression. Measurement of cytoplasmic mRNA levels demonstrated that the 
difference in protein expression is due to translational differences and 
not mRNA stability. 
Retroviruses in general do not show a similar preference towards A and T as 
found for HIV. But if this family was divided into two subgroups, 
lentiviruses and non-lentiviral retroviruses, a similar preference to A 
and, less frequently, T, was detected at the third codon position for 
lentiviruses. Thus, the availing evidence suggests that lentiviruses 
retain a characteristic pattern of envelope codons not because of an 
inherent advantage to the reverse transcription or replication of such 
residues, but rather for some reason peculiar to the physiology of that 
class of viruses. The major difference between lentiviruses and 
non-complex retroviruses are additional regulatory and non-essentially 
accessory genes in lentiviruses, as already mentioned. Thus, one simple 
explanation for the restriction of envelope expression might be that an 
important regulatory mechanism of one of these additional molecules is 
based on it. In fact, it is known that one of these proteins, rev, which 
most likely has homologues in all lentiviruses. Thus codon usage in viral 
mRNA is used to create a class of transcripts which is susceptible to the 
stimulatory action of rev. This hypothesis was proved using a similar 
strategy as above, but this time codon usage was changed into the inverse 
direction. Codon usage of a highly expressed cellular gene was substituted 
with the most frequently used codons in the HIV envelope. As assumed, 
expression levels were considerably lower in comparison to the native 
molecule, almost two orders of magnitude when analyzed by 
immunofluorescence of the surface expressed molecule (see 4.7). If rev was 
coexpressed in trans and an RRE element was present in cis only a slight 
induction was found for the surface molecule. However, if THY-1 was 
expressed as a secreted molecule, the induction by rev was much more 
prominent, supporting the above hypothesis. This can probably be explained 
by accumulation of secreted protein in the supernatant, which considerably 
amplifies the rev effect. If rev only induces a minor increase for surface 
molecules in general, induction of HIV envelope by rev cannot have the 
purpose of an increased surface abundance, but rather of an increased 
intracellular gp160 level. It is completely unclear at the moment why this 
should be the case. 
To test whether small subtotal elements of a gene are sufficient to 
restrict expression and render it rev-dependent rTHY1env:immunoglobulin 
fusion proteins were generated, in which only about one third of the total 
gene had the envelope codon usage. Expression levels of this construct 
were on an intermediate level, indicating that the rTHY-1env negative 
sequence element is not dominant over the immunoglobulin part. This fusion 
protein was not or only slightly rev-responsive, indicating that only 
genes almost completely suppressed can be rev-responsive. 
Another characteristic feature that was found in the codon frequency tables 
is a striking underrepresentation of CpG triplets. In a comparative study 
of codon usage in E. coli, yeast, drosophila and primates it was shown 
that in a high number of analyzed primate genes the 8 least used codons 
contain all codons with the CpG dinucleotide sequence. Avoidance of codons 
containing this dinucleotide motif was also found in the sequence of other 
retroviruses. It seems plausible that the reason for underrepresentation 
of CpG-bearing triplets has something to do with avoidance of gene 
silencing by methylation of CpG cytosines. The expected number of CpG 
dinucleotides for HIV as a whole is about one fifth that expected on the 
basis of the base composition. This might indicate that the possibility of 
high expression is restored, and that the gene in fact has to be highly 
expressed at some point during viral pathogenesis. 
The results presented herein clearly indicate that codon preference has a 
severe effect on protein levels, and suggest that translational elongation 
is controlling mammalian gene expression. However, other factors may play 
a role. First, abundance of not maximally loaded mRNA's in eukaryotic 
cells indicates that initiation is rate limiting for translation in at 
least some cases, since otherwise all transcripts would be completely 
covered by ribosomes. Furthermore, if ribosome stalling and subsequent 
mRNA degradation were the mechanism, suppression by rare codons could most 
likely not be reversed by any regulatory mechanism like the one presented 
herein. One possible explanation for the influence of both initiation and 
elongation on translational activity is that the rate of initiation, or 
access to ribosomes, is controlled in part by cues distributed throughout 
the RNA, such that the lentiviral codons predispose the RNA to accumulate 
in a pool of poorly initiated RNAs. However, this limitation need not be 
kinetic; for example, the choice of codons could influence the probability 
that a given translation product, once initiated, is properly completed. 
Under this mechanism, abundance of less favored codons would incur a 
significant cumulative probability of failure to complete the nascent 
polypeptide chain. The sequestered RNA would then be lent an improved rate 
of initiation by the action of rev. Since adenine residues are abundant in 
rev-responsive transcripts, it could be that RNA adenine methylation 
mediates this translational suppression. 
Detailed Procedures 
The following procedures were used in the above-described experiments. 
Sequence Analysis 
Sequence analyses employed the software developed by the University of 
Wisconsin Computer Group. 
Plasmid constructions 
Plasmid constructions employed the following methods. Vectors and insert 
DNA was digested at a concentration of 0.5 .mu.g/10 .mu.l in the 
appropriate restriction buffer for 1-4 hours (total reaction volume 
approximately 30 .mu.l). Digested vector was treated with 10% (v/v) of 1 
.mu.g/ml calf intestine alkaline phosphatase for 30 min prior to gel 
electrophoresis. Both vector and insert digests (5 to 10 .mu.l each) were 
run on a 1.5% low melting agarose gel with TAE buffer. Gel slices 
containing bands of interest were transferred into a 1.5 ml reaction tube, 
melted at 65.degree. C. and directly added to the ligation without removal 
of the agarose. Ligations were typically done in a total volume of 25 
.mu.l in 1.times. Low Buffer 1.times. Ligation Additions with 200-400 U of 
ligase, 1 .mu.l of vector, and 4 .mu.l of insert. When necessary, 5' 
overhanging ends were filled by adding 1/10 volume of 250 .mu.M dNTPs and 
2-5 U of Klenow polymerase to heat inactivated or phenol extracted digests 
and incubating for approximately 20 min at room temperature. When 
necessary, 3' overhanging ends were filled by adding 1/10 volume of 2.5 mM 
dNTPs and 5-10 U of T4 DNA polymerase to heat inactivated or phenol 
extracted digests, followed by incubation at 37.degree. C. for 30 min. The 
following buffers were used in these reactions: 10.times. Low buffer (60 
mM Tris HCl, pH 7.5, 60 mM MgCl.sub.2, 50 mM NaCl, 4 mg/ml BSA, 70 mM 
.beta.-mercaptoethanol, 0.02% NaN.sub.3); 10.times. Medium buffer (60 mM 
Tris HCl, pH 7.5, 60 mM MgCl.sub.2, 50 mM NaCl, 4 mg/ml BSA, 70 mM 
.beta.-mercaptoethanol, 0.02% NaN.sub.3); 10.times. High buffer (60 mM 
Tris HCl, pH 7.5, 60 mM MgCl.sub.2, 50 mM NaCl, 4 mg/ml BSA, 70 mM 
.beta.-mercaptoethanol, 0.02% NaN.sub.3); 10.times. Ligation additions (1 
mM ATP, 20 mM DTT, 1 mg/ml BSA, 10 mM spermidine); 50.times. TAE (2M Tris 
acetate, 50 mM EDTA). 
Oligonucleotide synthesis and purification 
Oligonucleotides were produced on a Milligen 8750 synthesizer (Millipore). 
The columns were eluted with 1 ml of 30% ammonium hydroxide, and the 
eluted oligonucleotides were deblocked at 55.degree. C. for 6 to 12 hours. 
After deblockiong, 150 .mu.l of oligonucleotide were precipitated with 
10.times. volume of unsaturated n-butanol in 1.5 ml reaction tubes, 
followed by centrifugation at 15,000 rpm in a microfuge. The pellet was 
washed with 70% ethanol and resuspended in 50 .mu.l of H.sub.2 O. The 
concentration was determined by measuring the optical density at 260 nm in 
a dilution of 1:333 (1 OD.sub.260 =30 .mu.g/ml). 
The following oligonucleotides were used for construction of the synthetic 
gp120 gene (all sequences shown in this text are in 5' to 3' direction). 
oligo 1 forward (Nhe1): cgc ggg cta gcc acc gag aag ctg (SEQ ID NO:1). 
oligo 1: acc gag aag ctg tgg gtg acc gtg tac tac ggc gtg ccc gtg tgg aag ag 
ag gcc acc acc acc ctg ttc tgc gcc agc gac gcc aag gcg tac gac acc gag gtg 
cac aac gtg tgg gcc acc cag gcg tgc gtg ccc acc gac ccc aac ccc cag gag 
gtg gag ctc gtg aacgtg acc gag aac ttc aac atg (SEQ ID NO:2). 
oligo 1 reverse: cca cca tgt tgt tct tcc aca tgt tga agt tct c (SEQ ID 
NO:3). 
oligo 2 forward: gac cga gaa ctt caa cat gtg gaa gaa caa cat (SEQ ID NO:4) 
oligo 2: tgg aag aac aac atg gtg gag cag atg cat gag gac atc atc agc ctg 
tgg gac cag agc ctg aag ccc tgc gtg aag ctg acc cc ctg tgc gtg acc tg aac 
tgc acc gac ctg agg aac acc acc aac acc aac ac agc acc gcc aac aac aac agc 
aac agc gag ggc acc atc aag ggc ggc gag atg (SEQ ID NO:5). 
oligo 2 reverse (Pst1): gtt gaa gct gca gtt ctt cat ctc gcc gcc ctt (SEQ ID 
NO:6). 
oligo 3 forward (Pst1): gaa gaa ctg cag ctt caa cat cac cac cag c (SEQ ID 
NO:7). 
oligo 3: aac atc acc acc agc atc cgc gac aag atg cag aag gag tac gcc ctg 
ctg tac aag ctg gat atc gtg agc atc gac aac gac agc acc agc tac cgc ctg 
atc tcc tgc aac acc agc gtg atc acc cag gcc tgc ccc aag atc agc ttc gag 
ccc atc ccc atc cac tac tgc gcc ccc gcc ggc ttc gcc (SEQ ID NO:8). 
oligo 3 reverse: gaa ctt ctt gtc ggc ggc gaa gcc ggc ggg (SEQ ID NO:9). 
oligo 4 forward: gcg ccc ccg ccg gct tcg cca tcc tga agt gca acg aca aga 
agt tc (SEQ ID NO:10) 
oligo 4: gcc gac aag aag ttc agc ggc aag ggc agc tgc aag aac gtg agc acc 
gtg cag tgc acc cac ggc atc cgg ccg gtg gtg agc acc cag ctc ctg ctg aac 
ggc agc ctg gcc gag gag gag gtg gtg atc cgc agc gag aac ttc acc gac aac 
gcc aag acc atc atc gtg cac ctg aat gag agc gtg cag atc (SEQ ID NO:11) 
oligo 4 reverse (Mlu1): agt tgg gac gcg tgc agt tga tct gca cgc tct c (SEQ 
ID NO:12). 
oligo 5 forward (Mlu1): gag agc gtg cag atc aac tgc acg cgt ccc (SEQ ID 
NO:13). 
oligo 5: aac tgc acg cgt ccc aac tac aac aag cgc aag cgc atc cac atc ggc 
ccc ggg cgc gcc ttc tac acc acc aag aac atc atc ggc acc atc ctc cag gcc 
cac tgc aac atc tct aga (SEQ ID NO:14). 
oligo 5 reverse: gtc gtt cca ctt ggc tct aga gat gtt gca (SEQ ID NO:15). 
oligo 6 forward: gca aca tct cta gag cca agt gga acg ac (SEQ ID NO:16). 
oligo 6: gcc aag tgg aac gac acc ctg cgc cag atc gtg agc aag ctg aag gag 
cag ttc aag aac aag acc atc gtg ttc ac cag agc agc ggc ggc gac ccc gag atc 
gtg atg cac agc ttc aac tgc ggc ggc (SEQ ID NO:17). 
oligo 6 reverse (EcoR1): gca gta gaa gaa ttc gcc gcc gca gtt ga (SEQ ID 
NO:18). 
oligo 7 forward (EcoR1): tca act gcg gcg gcg aat tct tct act gc (SEQ ID 
NO:19). 
oligo 7: ggc gaa ttc ttc tac tgc aac acc agc ccc ctg ttc aac agc acc tgg 
aac ggc aac aac acc tgg aac aac acc acc ggc agc aac aac aat att acc ctc 
cag tgc aag atc aag cag atc atc aac atg tgg cag gag gtg ggc aag gcc atg 
tac gcc ccc ccc atc gag ggc cag atc cgg tgc agc agc (SEQ ID NO:20) 
oligo 7 reverse: gca gac cgg tga tgt tgc tgc tgc acc gga tct ggc cct c (SEQ 
ID NO:21). 
oligo 8 forward: cga ggg cca gat ccg gtg cag cag caa cat cac cgg tct g (SEQ 
ID NO:22). 
oligo 8: aac atc acc ggt ctg ctg ctg acc cgc gac ggc ggc aag gac acc gac 
acc aac gac acc gaa atc ttc cgc ccc ggc ggc ggc gac atg cgc gac aac tgg 
aga tct gag ctg tac aag tac aag gtg gtg acg atc gag ccc ctg ggc gtg gcc 
ccc acc aag gcc aag cgc cgc gtg gtg cag cgc gag aag cgc (SEQ ID NO:23). 
oligo 8 reverse (Not1): cgc ggg cgg ccg ctt tag cgc ttc tcg cgc tgc acc ac 
(SEQ ID NO:24). 
The following oligonucleotides were used for the construction of the 
ratTHY-1env gene. 
oligo 1 forward (BamH1/Hind3): cgc ggg gga tcc aag ctt acc atg att cca gta 
ata agt (SEQ ID NO:25). 
oligo 1: atg aat cca gta ata agt ata aca tta tta tta agt gta tta caa atg 
agt aga gga caa aga gta ata agt tta aca gca tct tta gta aat caa aat ttg 
aga tta gat tgt aga cat gaa aat aat aca aat ttg cca ata caa cat gaa ttt 
tca tta acg (SEQ ID NO:26). 
oligo 1 reverse (EcoR1/Mlu1): cgc ggg gaa ttc acg cgt taa tga aaa ttc atg 
ttg (SEQ ID NO:27). 
oligo 2 forward (BamH1/Mlu1): cgc gga tcc acg cgt gaa aaa aaa aaa cat (SEQ 
ID NO:28). 
oligo 2: cgt gaa aaa aaa aaa cat gta tta agt gga aca tta gga gta cca gaa 
cat aca tat aga agt aga gta aat ttg ttt agt gat aga ttc ata aaa gta tta 
aca tta gca aat ttt aca aca aaa gat gaa gga gat tat atg tgt gag (SEQ ID 
NO:29). 
oligo 2 reverse (EcoR1/Sac1): cgc gaa ttc gag ctc aca cat ata atc tcc (SEQ 
ID NO:30). 
oligo 3 forward (BamH1/Sac1): cgc gga tcc gag ctc aga gta agt gga caa (SEQ 
ID NO:31). 
oligo 3: ctc aga gta agt gga caa aat cca aca agt agt aat aaa aca ata aat 
gta ata aga gat aaa tta gta aaa tgt ga gga ata agt tta tta gta caa aat aca 
agt tgg tta tta tta tta tta tta agt tta agt ttt tta caa gca aca gat ttt 
ata agt tta tga (SEQ ID NO:32). 
oligo 3 reverse (EcoR1/Not1): cgc gaa ttc gcg gcc gct tca taa act tat aaa 
atc (SEQ ID NO:33). 
Polymerase Chain Reaction 
Short, overlapping 15 to 25 mer oligonucleotides annealing at both ends 
were used to amplify the long oligonuclotides by polymerase chain reaction 
(PCR). Typical PCR conditions were: 35 cycles, 55.degree. C. annealing 
temperature, 0.2 sec extension time. PCR products were gel purified, 
phenol extracted, and used in a subsequent PCR to generate longer 
fragments consisting of two adjacent small fragments. These longer 
fragments were cloned into a CDM7-derived plasmid containing a leader 
sequence of the CD5 surface molecule followed by a 
Nhe1/Pst1/Mlu1/EcoR1/BamH1 polylinker. 
The following solutions were used in these reactions: 10.times. PCR buffer 
(500 mM KCl, 100 mM Tris HCl, pH 7.5, 8 mM MgCl.sub.2, 2 mM each dNTP). 
The final buffer was complemented with 10% DMSO to increase fidelity of 
the Taq polymerase. 
Small scale DNA preparation 
Transformed bacteria were grown in 3 ml LB cultures for more than 6 hours 
or overnight. Approximately 1.5 ml of each culture was poured into 1.5 ml 
microfuge tubes, spun for 20 seconds to pellet cells and resuspended in 
200 .mu.l of solution I. Subsequently 400 .mu.l of solution II and 300 
.mu.l of solution III were added. The microfuge tubes were capped, mixed 
and spun for &gt;30 sec. Supernatants were transferred into fresh tubes and 
phenol extracted once. DNA was precipitated by filling the tubes with 
isopropanol, mixing, and spinning in a microfuge for &gt;2 min. The pellets 
were rinsed in 70 % ethanol and resuspended in 50 .mu.l dH.sub.2 O 
containing 10 .mu.l of RNAse A. The following media and solutions were 
used in these procedures: LB medium (1.0% NaCl, 0.5% yeast extract, 1.0% 
trypton); solution I (10 mM EDTA pH 8.0); solution II (0.2M NaOH, 1.0% 
SDS); solution III (2.5M KOAc, 2.5M glacial aceatic acid); phenol (pH 
adjusted to 6.0, overlaid with TE); TE (10 mM Tris HCl, pH 7.5, 1 mM EDTA 
pH 8.0). 
Large scale DNA preparation 
One liter cultures of transformed bacteria were grown 24 to 36 hours 
(MC1061p3 transformed with pCDM derivatives) or 12 to 16 hours (MC1061 
transformed with pUC derivatives) at 37.degree. C. in either M9 bacterial 
medium (pCDM derivatives) or LB (pUC derivatives). Bacteria were spun down 
in 1 liter bottles using a Beckman J6 centrifuge at 4,200 rpm for 20 min. 
The pellet was resuspended in 40 ml of solution I. Subsequently, 80 ml of 
solution II and 40 ml of solution III were added and the bottles were 
shaken semivigorously until lumps of 2 to 3 mm size developed. The bottle 
was spun at 4,200 rpm for 5 min and the supernatant was poured through 
cheesecloth into a 250 ml bottle. Isopropanol was added to the top and the 
bottle was spun at 4,200 rpm for 10 min. The pellet was resuspended in 4.1 
ml of solution I and added to 4.5 g of cesium chloride, 0.3 ml of 10 mg/ml 
ethidium bromide, and 0.1 ml of 1% Triton X100 solution. The tubes were 
spun in a Beckman J2 high speed centrifuge at 10,000 rpm for 5 min. The 
supernatant was transferred into Beckman Quick Seal ultracentrifuge tubes, 
which were then sealed and spun in a Beckman ultracentrifuge using a NVT90 
fixed angle rotor at 80,000 rpm for &gt;2.5 hours. The band was extracted by 
visible light using a 1 ml syringe and 20 gauge needle. An equal volume of 
dH.sub.2 O was added to the extracted material. DNA was extracted once 
with n-butanol saturated with 1M sodium chloride, followed by addition of 
an equal volume of 10M ammonium acetate/1 mM EDTA. The material was poured 
into a 13 ml snap tube which was than filled to the top with absolute 
ethanol, mixed, and spun in a Beckman J2 centrifuge at 10,000 rpm for 10 
min. The pellet was rinsed with 70% ethanol and resuspended in 0.5 to 1 ml 
of H.sub.2 O. The DNA concentration was determined by measuring the 
optical density at 260 nm in a dilution of 1:200 (1 OD.sub.260 =50 
.mu.g/ml). 
The following media and buffers were used in these procedures: M9 bacterial 
medium (10 g M9 salts, 10 g casamino acids (hydrolysed), 10 ml M9 
additions, 7.5 .mu.g/ml tetracycline (500 .mu.l of a 15 mg/ml stock 
solution), 12.5 .mu.g/ml ampicillin (125 .mu.l of a 10 mg/ml stock 
solution); M9 additions (10 mM CaCl.sub.2, 100 mM MgSO.sub.4, 200 .mu.g/ml 
thiamine, 70% glycerol); LB medium (1.0% NaCl, 0.5% yeast extract, 1.0% 
trypton); Solution I (10 mM EDTA pH 8.0); Solution II (0.2M NaOH 1.0% 
SDS); Solution III (2.5M KOAC 2.5M HOAc) 
Sequencing 
Synthetic genes were sequenced by the Sanger dideoxynucleotide method. In 
brief, 20 to 50 .mu.g double-stranded plasmid DNA were denatured in 0.5M 
NaOH for 5 min. Subsequently the DNA was precipitated with 1/10 volume of 
sodium acetate (pH 5.2) and 2 volumes of ethanol and centrifuged for 5 
min. The pellet was washed with 70% ethanol and resuspended at a 
concentration of 1 .mu.g/.mu.l. The annealing reaction was carried out 
with 4 .mu.g of template DNA and 40 ng of primer in 1.times. annealing 
buffer in a final volume of 10 .mu.l. The reaction was heated to 
65.degree. C. and slowly cooled to 37.degree. C. In a separate tube 1 
.mu.l of 0.1M DTT, 2 .mu.l of labeling mix, 0.75 .mu.l of dH.sub.2 O, 1 
.mu.l of .sup.35 S! dATP (10 .mu.Ci), and 0.25 .mu.l of Sequenase.TM. (12 
U/.mu.l) were added for each reaction. Five .mu.l of this mix were added 
to each annealed primer-template tube and incubated for 5 min at room 
temperature. For each labeling reaction 2.5 .mu.l of each of the 4 
termination mixes were added on a Terasaki plate and prewarmed at 
37.degree. C. At the end of the incubation period 3.5 .mu.l of labeling 
reaction were added to each of the 4 termination mixes. After 5 min, 4 
.mu.l of stop solution were added to each reaction and the Terasaki plate 
was incubated at 80.degree. C. for 10 min in an oven. The sequencing 
reactions were run on 5% denaturing polyacrylamide gel. An acrylamide 
solution was prepared by adding 200 ml of 10.times. TBE buffer and 957 ml 
of dH.sub.2 O to 100 g of acrylamide:bisacrylamide (29:1). 5% 
polyacrylamide 46% urea and 1.times. TBE gel was prepared by combining 38 
ml of acrylamide solution and 28 g urea. Polymerization was initiated by 
the addition of 400 .mu.l of 10% ammonium peroxodisulfate and 60 .mu.l of 
TEMED. Gels were poured using silanized glass plates and sharktooth combs 
and run in 1.times. TBE buffer at 60 to 100 W for 2 to 4 hours (depending 
on the region to be read). Gels were transferred to Whatman blotting 
paper, dried at 80.degree. C. for about 1 hour, and exposed to x-ray film 
at room temperature. Typically exposure time was 12 hours. The following 
solutions were used in these procedures: 5.times. Annealing buffer (200 mM 
Tris HCl, pH 7.5, 100 mM MgCl.sub.2, 250 mM NaCl); Labelling Mix (7.5 
.mu.M each dCTP, dGTP, and dTTP); Termination Mixes (80 .mu.M each dNTP, 
50 mM NaCl, 8 .mu.M ddNTP (one each)); Stop solution (95% formamide, 20 mM 
EDTA, 0.05% bromphenol blue, 0.05% xylencyanol); 5.times. TBE (0.9M Tris 
borate, 20 mM EDTA); Polyacrylamide solution (96.7 g polyacrylamide, 3.3 g 
bisacrylamide, 200 ml 1.times. TBE, 957 ml dH.sub.2 O). 
RNA isolation 
Cytoplasmic RNA was isolated from calcium phosphate transfected 293T cells 
36 hours post transfection and from vaccinia infected Hela cells 16 hours 
post infection essentially as described by Gilman. (Gilman Preparation of 
cytoplasmic RNA from tissue culture cells. In Current Protocols in 
Molecular Biology, Ausubel et al, eds., Wiley & Sons, New York, 1992). 
Briefly, cells were lysed in 400 .mu.l lysis buffer, nuclei were spun out, 
and SDS and proteinase K were added to 0.2% and 0.2 mg/ml respectively. 
The cytoplasmic extracts were incubated at 37.degree. C. for 20 min, 
phenol/chloroform extracted twice, and precipitated. The RNA was dissolved 
in 100 .mu.l buffer I and incubated at 37.degree. C. for 20 min. The 
reaction was stopped by adding 25 .mu.l stop buffer and precipitated 
again. 
The following solutions were used in this procedure: Lysis Buffer (TE 
containing with 50 mM Tris pH 8.0, 100 mM NaCl, 5 mM MgCl.sub.2, 0.5% 
NP40); Buffer I (TE buffer with 10 mM MgCl.sub.2, 1 mM DTT, 0.5 U/.mu.l 
placental RNAse inhibitor, 0.1 U/.mu.l RNAse free DNAse I); Stop buffer 
(50 mM EDTA 1.5M NaOAc 1.0% SDS). 
Slot blot analysis 
For slot blot analysis 10 .mu.g of cytoplasmic RNA was dissolved in 50 
.mu.l dH.sub.2 O to which 150 .mu.l of 10.times. SSC/18% formaldehyde were 
added. The solubilized RNA was then incubated at 65.degree. C. for 15 min 
and spotted onto with a slot blot apparatus. Radioactively labelled probes 
of 1.5 kb gp120IIIb and syngp120mn fragments were used for hybridization. 
Each of the two fragments was random labelled in a 50 .mu.l reaction with 
10 .mu.l of 5.times. oligo-labelling buffer, 8 .mu.l of 2.5 mg/ml BSA, 4 
.mu.l of .alpha..sup.32 P!-dCTP (20 uCi/.mu.l; 6000 Ci/mmol), and 5 U of 
Klenow fragment. After 1 to 3 hours incubation at 37.degree. C. 100 .mu.l 
of TE were added and unincorporated .alpha..sup.32 P!-dCTP was eliminated 
using a G50 spin column. Activity was measured in a Beckman beta-counter, 
and equal specific activities were used for hybridization. Membranes were 
pre-hybridized for 2 hours and hybridized for 12 to 24 hours at 42.degree. 
C. with 0.5.times.10.sup.6 cpm probe per ml hybridization fluid. The 
membrane was washed twice (5 min) with washing buffer I at room 
temperature, for one hour in washing buffer II at 65.degree. C., and then 
exposed to x-ray film. Similar results were obtained using a 1.1 kb 
Not1/Sfi1 fragment of pCDM7 containing the 3' untranslated region. Control 
hybridizations were done in parallel with a random-labelled human 
beta-actin probe. RNA expression was quantitated by scanning the 
hybridized nitrocellulose membranes with a Magnetic Dynamics 
phosphorimager. 
The following solutions were used in this procedure: 5.times. 
Oligo-labelling buffer (250 mM Tris HCl, pH 8.0, 25 mM MgCl.sub.2, 5 mM 
.beta.-mercaptoethanol, 2 mM dATP, 2 mM dGTP, 2 mM dTTP, 1M Hepes pH 6.6, 
1 mg/ml hexanucleotides dNTP!6); Hybridization Solution (.sub.-- M sodium 
phosphate, 250 mM NaCl, 7% SDS, 1 mM EDTA, 5% dextrane sulfate, 50% 
formamide, 100 .mu.g/ml denatured salmon sperm DNA); Washing buffer I 
(2.times. SSC, 0.1% SDS); Washing buffer II (0.5.times. SSC, 0.1% SDS); 
20.times. SSC (3M NaCl, 0.3M Na.sub.3 citrate, pH adjusted to 7.0). 
Vaccinia recombination 
Vaccinia recombination used a modification of the method described by Romeo 
and Seed (Romeo and Seed, Cell, 64: 1037, 1991). Briefly, CV1 cells at 70 
to 90% confluency were infected with 1 to 3 .mu.l of a wildtype vaccinia 
stock WR (2.times.10.sup.8 pfu/ml) for 1 hour in culture medium without 
calf serum. After 24 hours, the cells were transfected by calcium 
phosphate with 25 .mu.g TKG plasmid DNA per dish. After an additional 24 
to 48 hours the cells were scraped off the plate, spun down, and 
resuspended in a volume of 1 ml. After 3 freeze/thaw cycles trypsin was 
added to 0.05 mg/ml and lysates were incubated for 20 min. A dilution 
series of 10, 1 and 0.1 .mu.l of this lysate was used to infect small 
dishes (6 cm) of CV1 cells, that had been pretreated with 12.5 .mu.g/ml 
mycophenolic acid, 0.25 mg/ml xanthin and 1.36 mg/ml hypoxanthine for 6 
hours. Infected cells were cultured for 2 to 3 days, and subsequently 
stained with the monoclonal antibody NEA9301 against gp120 and an alkaline 
phosphatase conjugated secondary antibody. Cells were incubated with 0.33 
mg/ml NBT and 0.16 mg/ml BCIP in AP-buffer and finally overlaid with 1% 
agarose in PBS. Positive plaques were picked and resuspended in 100 .mu.l 
Tris pH 9.0. The plaque purification was repeated once. To produce high 
titer stocks the infection was slowly scaled up. Finally, one large plate 
of Hela cells was infected with half of the virus of the previous round. 
Infected cells were detached in 3 ml of PBS, lysed with a Dounce 
homogenizer and cleared from larger debris by centrifugation. VPE-8 
recombinant vaccinia stocks were kindly provided by the AIDS repository, 
Rockville, Md., and express HIV-1 IIIB gp120 under the 7.5 mixed 
early/late promoter (Earl et al., J. Virol., 65:31, 1991). In all 
experiments with recombinant vaccina cells were infected at a multiplicity 
of infection of at least 10. 
The following solution was used in this procedure: AP buffer (100 mM Tris 
HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl.sub.2) 
Cell culture 
The monkey kidney carcinoma cell lines CV1 and Cos7, the human kidney 
carcinoma cell line 293T, and the human cervix carcinoma cell line Hela 
were obtained from the American Tissue Typing Collection and were 
maintained in supplemented IMDM. They were kept on 10 cm tissue culture 
plates and typically split 1:5 to 1:20 every 3 to 4 days. 
The following medium was used in this procedure: Supplemented IMDM (90% 
Iscove's modified Dulbecco Medium, 10% calf serum, iron-complemented, heat 
inactivated 30 min 56.degree. C., 0.3 mg/ml L-glutamine, 25 .mu.g/ml 
gentamycin 0.5 mM .beta.-mercaptoethanol (pH adjusted with 5M NaOH, 0.5 
ml)). 
Transfection 
Calcium phosphate transfection of 293T cells was performed by slowly adding 
and under vortexing 10 .mu.g plasmid DNA in 250 .mu.l 0.25M CaCl.sub.2 to 
the same volume of 2.times. HEBS buffer while vortexing. After incubation 
for 10 to 30 min at room temperature the DNA precipitate was added to a 
small dish of 50 to 70% confluent cells. In cotransfection experiments 
with rev, cells were transfected with 10 .mu.g gp120IIIb, gp120IIIbrre, 
syngp120mnrre or rTHY-1enveg1rre and 10 .mu.g of pCMVrev or CDM7 plasmid 
DNA. 
The following solutions were used in this procedure: 2.times. HEBS buffer 
(280 mM NaCl, 10 mM KCl, 1.5 mM sterile filtered); 0.25 mM CaCl.sub.2 
(autoclaved). 
Immunoprecipitation 
After 48 to 60 hours medium was exchanged and cells were incubated for 
additional 12 hours in Cys/Met-free medium containing 200 .mu.Ci of 
.sup.35 S-translabel. Supernatants were harvested and spun for 15 min at 
3000 rpm to remove debris. After addition of protease inhibitors 
leupeptin, aprotinin and PMSF to 2.5 .mu.g/ml, 50 .mu.g/ml, 100 .mu.g/ml 
respectively, 1 ml of supernatant was incubated with either 10 .mu.l of 
packed protein A sepharose alone (rTHY-1enveg1rre) or with protein A 
sepharose and 3 .mu.g of a purified CD4/immunoglobulin fusion protein 
(kindly provided by Behring) (all gp120 constructs) at 4.degree. C. for 12 
hours on a rotator. Subsequently the protein A beads were washed 5 times 
for 5 to 15 min each time. After the final wash 10 .mu.l of loading buffer 
containing was added, samples were boiled for 3 min and applied on 7% (all 
gp120 constructs) or 10% (rTHY-1enveg1rre) SDS polyacrylamide gels (Tris 
pH 8.8 buffer in the resolving, Tris pH 6.8 buffer in the stacking gel, 
Tris-glycine running buffer, Maniatis et al. 1989). Gels were fixed in 10% 
acetic acid and 10% methanol, incubated with Amplify for 20 min, dried and 
exposed for 12 hours. 
The following buffers and solutions were used in this procedure: Wash 
buffer (100 mM Tris, pH 7.5, 150 mM NaCl, 5 mM CaCl.sub.2, 1% NP-40); 
5.times. Running Buffer (125 mM Tris, 1.25M Glycine, 0.5% SDS); Loading 
buffer (10% glycerol, 4% SDS, 4% .beta.-mercaptoethanol, 0.02% bromphenol 
blue). 
Immunofluorescence 
293T cells were transfected by calcium phosphate coprecipitation and 
analyzed for surface THY-1 expression after 3 days. After detachment with 
1 mM EDTA/PBS, cells were stained with the monoclonal antibody OX-7 in a 
dilution of 1:250 at 4.degree. C. for 20 min, washed with PBS and 
subsequently incubated with a 1:500 dilution of a FITC-conjugated goat 
anti-mouse immunoglobulin antiserum. Cells were washed again, resuspended 
in 0.5 ml of a fixing solution, and analyzed on an EPICS XL 
cytofluorometer (Coulter). 
The following solutions were used in this procedure: PBS (137 mM NaCl, 2.7 
mM KCl, 4.3 mM Na.sub.2 HPO.sub.4, 1.4 mM KH.sub.2 PO.sub.4, pH adjusted 
to 7.4); Fixing solution (2% formaldehyde in PBS). 
ELISA 
The concentration of gp120 in culture supernatants was determined using 
CD4-coated ELISA plates and goat anti-gp120 antisera in the soluble phase. 
Supernatants of 293T cells transfected by calcium phosphate were harvested 
after 4 days, spun at 3000 rpm for 10 min to remove debris and incubated 
for 12 hours at 4.degree. C. on the plates. After 6 washes with PBS 100 
.mu.l of goat anti-gp120 antisera diluted 1:200 were added for 2 hours. 
The plates were washed again and incubated for 2 hours with a 
peroxidase-conjugated rabbit anti-goat IgG antiserum 1:1000. Subsequently 
the plates were washed and incubated for 30 min with 100 .mu.l of 
substrate solution containing 2 mg/ml o-phenylenediamine in sodium citrate 
buffer. The reaction was finally stopped with 100 .mu.l of 4M sulfuric 
acid. Plates were read at 490 nm with a Coulter microplate reader. 
Purified recombinant gp120IIIb was used as a control. The following 
buffers and solutions were used in this procedure: Wash buffer (0.1% NP40 
in PBS); Substrate solution (2 mg/ml o-phenylenediamine in sodium citrate 
buffer). 
Use 
The synthetic genes of the invention are useful for expressing a protein 
normally expressed in mammalian cells in cell culture (e.g. for commercial 
production of human proteins such as hGH, TPA, Factor VII, and Factor IX). 
The synthetic genes of the invention are also useful for gene therapy. 
__________________________________________________________________________ 
SEQUENCE LISTING 
(1) GENERAL INFORMATION: 
(iii) NUMBER OF SEQUENCES: 37 
(2) INFORMATION FOR SEQ ID NO:1: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
CGCGGGCTAGCCACCGAGAAGCTG24 
(2) INFORMATION FOR SEQ ID NO:2: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 196 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
ACCGAGAAGCTGTGGGTGACCGTGTACTACGGCGTGCCCGTGTGGAAGAGAGGCCACCAC60 
CACCCTGTTCTGCGCCAGCGACGCCAAGGCGTACGACACCGAGGTGCACAACGTGTGGGC120 
CACCCAGGCGTGCGTGCCCACCGACCCCAACCCCCAGGAGGTGGAGCTCGTGAACGTGAC180 
CGAGAACTTCAACATG196 
(2) INFORMATION FOR SEQ ID NO:3: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
CCACCATGTTGTTCTTCCACATGTTGAAGTTCTC34 
(2) INFORMATION FOR SEQ ID NO:4: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
GACCGAGAACTTCAACATGTGGAAGAACAACAT33 
(2) INFORMATION FOR SEQ ID NO:5: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 192 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
TGGAAGAACAACATGGTGGAGCAGATGCATGAGGACATCATCAGCCTGTGGGACCAGAGC60 
CTGAAGCCCTGCGTGAAGCTGACCCCCTGTGCGTGACCTGAACTGCACCGACCTGAGGAA120 
CACCACCAACACCAACACAGCACCGCCAACAACAACAGCAACAGCGAGGGCACCATCAAG180 
GGCGGCGAGATG192 
(2) INFORMATION FOR SEQ ID NO:6: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 33 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
GTTGAAGCTGCAGTTCTTCATCTCGCCGCCCTT33 
(2) INFORMATION FOR SEQ ID NO:7: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 31 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
GAAGAACTGCAGCTTCAACATCACCACCAGC31 
(2) INFORMATION FOR SEQ ID NO:8: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 195 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
AACATCACCACCAGCATCCGCGACAAGATGCAGAAGGAGTACGCCCTGCTGTACAAGCTG60 
GATATCGTGAGCATCGACAACGACAGCACCAGCTACCGCCTGATCTCCTGCAACACCAGC120 
GTGATCACCCAGGCCTGCCCCAAGATCAGCTTCGAGCCCATCCCCATCCACTACTGCGCC180 
CCCGCCGGCTTCGCC195 
(2) INFORMATION FOR SEQ ID NO:9: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
GAACTTCTTGTCGGCGGCGAAGCCGGCGGG30 
(2) INFORMATION FOR SEQ ID NO:10: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 47 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
GCGCCCCCGCCGGCTTCGCCATCCTGAAGTGCAACGACAAGAAGTTC47 
(2) INFORMATION FOR SEQ ID NO:11: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 198 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
GCCGACAAGAAGTTCAGCGGCAAGGGCAGCTGCAAGAACGTGAGCACCGTGCAGTGCACC60 
CACGGCATCCGGCCGGTGGTGAGCACCCAGCTCCTGCTGAACGGCAGCCTGGCCGAGGAG120 
GAGGTGGTGATCCGCAGCGAGAACTTCACCGACAACGCCAAGACCATCATCGTGCACCTG180 
AATGAGAGCGTGCAGATC198 
(2) INFORMATION FOR SEQ ID NO:12: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 34 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
AGTTGGGACGCGTGCAGTTGATCTGCACGCTCTC34 
(2) INFORMATION FOR SEQ ID NO:13: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: 
GAGAGCGTGCAGATCAACTGCACGCGTCCC30 
(2) INFORMATION FOR SEQ ID NO:14: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 120 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: 
AACTGCACGCGTCCCAACTACAACAAGCGCAAGCGCATCCACATCGGCCCCGGGCGCGCC60 
TTCTACACCACCAAGAACATCATCGGCACCATCCTCCAGGCCCACTGCAACATCTCTAGA120 
(2) INFORMATION FOR SEQ ID NO:15: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:15: 
GTCGTTCCACTTGGCTCTAGAGATGTTGCA30 
(2) INFORMATION FOR SEQ ID NO:16: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:16: 
GCAACATCTCTAGAGCCAAGTGGAACGAC29 
(2) INFORMATION FOR SEQ ID NO:17: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 131 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:17: 
GCCAAGTGGAACGACACCCTGCGCCAGATCGTGAGCAAGCTGAAGGAGCAGTTCAAGAAC60 
AAGACCATCGTGTTCACCAGAGCAGCGGCGGCGACCCCGAGATCGTGATGCACAGCTTCA120 
ACTGCGGCGGC131 
(2) INFORMATION FOR SEQ ID NO:18: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:18: 
GCAGTAGAAGAATTCGCCGCCGCAGTTGA29 
(2) INFORMATION FOR SEQ ID NO:19: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 29 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:19: 
TCAACTGCGGCGGCGAATTCTTCTACTGC29 
(2) INFORMATION FOR SEQ ID NO:20: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 195 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:20: 
GGCGAATTCTTCTACTGCAACACCAGCCCCCTGTTCAACAGCACCTGGAACGGCAACAAC60 
ACCTGGAACAACACCACCGGCAGCAACAACAATATTACCCTCCAGTGCAAGATCAAGCAG120 
ATCATCAACATGTGGCAGGAGGTGGGCAAGGCCATGTACGCCCCCCCCATCGAGGGCCAG180 
ATCCGGTGCAGCAGC195 
(2) INFORMATION FOR SEQ ID NO:21: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:21: 
GCAGACCGGTGATGTTGCTGCTGCACCGGATCTGGCCCTC40 
(2) INFORMATION FOR SEQ ID NO:22: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 40 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:22: 
CGAGGGCCAGATCCGGTGCAGCAGCAACATCACCGGTCTG40 
(2) INFORMATION FOR SEQ ID NO:23: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 242 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:23: 
AACATCACCGGTCTGCTGCTGCTGCTGACCCGGACGGCGGCAAGGACACCGACACCAACG60 
ACACCGAAATCTTCCGCGACGGCGGCAAGGACACCAACGACACCGAAATCTTCCGCCCCG120 
GCGGCGGCGACATGCGCGACAACTGGAGATCTGAGCTGTACAAGTACAAGGTGGTGACGA180 
TCGAGCCCCTGGGCGTGGCCCCCACCAAGGCCAAGCGCGCGGTGGTGCAGCGCGAGAAGC240 
GC242 
(2) INFORMATION FOR SEQ ID NO:24: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 38 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:24: 
CGCGGGCGGCCGCTTTAGCGCTTCTCGCGCTGCACCAC38 
(2) INFORMATION FOR SEQ ID NO:25: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 39 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:25: 
CGCGGGGGATCCAAGCTTACCATGATTCCAGTAATAAGT39 
(2) INFORMATION FOR SEQ ID NO:26: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 165 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:26: 
ATGAATCCAGTAATAAGTATAACATTATTATTAAGTGTATTACAAATGAGTAGAGGACAA60 
AGAGTAATAAGTTTAACAGCATCTTTAGTAAATCAAAATTTGAGATTAGATTGTAGACAT120 
GAAAATAATACAAATTTGCCAATACAACATGAATTTTCATTAACG165 
(2) INFORMATION FOR SEQ ID NO:27: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:27: 
CGCGGGGAATTCACGCGTTAATGAAAATTCATGTTG36 
(2) INFORMATION FOR SEQ ID NO:28: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:28: 
CGCGGATCCACGCGTGAAAAAAAAAAACAT30 
(2) INFORMATION FOR SEQ ID NO:29: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 149 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:29: 
CGTGAAAAAAAAAAACATGTATTAAGTGGAACATTAGGAGTACCAGAACATACATATAGA60 
AGTAGAGTAATTTGTTTAGTGATAGATTCATAAAAGTATTAACATTAGCAAATTTTACAA120 
CAAAAGATGAAGGAGATTATATGTGTGAG149 
(2) INFORMATION FOR SEQ ID NO:30: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:30: 
CGCGAATTCGAGCTCACACATATAATCTCC30 
(2) INFORMATION FOR SEQ ID NO:31: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:31: 
CGCGGATCCGAGCTCAGAGTAAGTGGACAA30 
(2) INFORMATION FOR SEQ ID NO:32: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 170 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:32: 
CTCAGAGTAAGTGGACAAAATCCAACAAGTAGTAATAAAACAATAAATGTAATAAGAGAT60 
AAATTAGTAAAATGTGAGGAATAAGTTTATTAGTACAAAATACAAGTTGGTTATTATTAT120 
TATTATTAAGTTTAAGTTTTTTACAAGCAACAGATTTTATAAGTTTATGA170 
(2) INFORMATION FOR SEQ ID NO:33: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 36 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:33: 
CGCGAATTCGCGGCCGCTTCATAAACTTATAAAATC36 
(2) INFORMATION FOR SEQ ID NO:34: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 1632 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:34: 
CTCGAGATCCATTGTGCTCTAAAGGAGATACCCGGCCAGACACCCTCACCTGCGGTGCCC60 
AGCTGCCCAGGCTGAGGCAAGAGAAGGCCAGAAACCATGCCCATGGGGTCTCTGCAACCG120 
CTGGCCACCTTGTACCTGCTGGGGATGCTGGTCGCTTCCGTGCTAGCCACCGAGAAGCTG180 
TGGGTGACCGTGTACTACGGCGTGCCCGTGTGGAAGGAGGCCACCACCACCCTGTTCTGC240 
GCCAGCGACGCCAAGGCGTACGACACCGAGGTGCACAACGTGTGGGCCACCCAGGCGTGC300 
GTGCCCACCGACCCCAACCCCCAGGAGGTGGAGCTCGTGAACGTGACCGAGAACTTCAAC360 
ATGTGGAAGAACAACATGGTGGAGCAGATGCATGAGGACATCATCAGCCTGTGGGACCAG420 
AGCCTGAAGCCCTGCGTGAAGCTGACCCCCCTGTGCGTGACCCTGAACTGCACCGACCTG480 
AGGAACACCACCAACACCAACAACAGCACCGCCAACAACAACAGCAACAGCGAGGGCACC540 
ATCAAGGGCGGCGAGATGAACAACTGCAGCTTCAACATCACCACCAGCATCCGCGACAAG600 
ATGCAGAAGGAGTACGCCCTGCTGTACAAGCTGGATATCGTGAGCATCGACAACGACAGC660 
ACCAGCTACCGCCTGATCTCCTGCAACACCAGCGTGATCACCCAGGCCTGGCCCAAGATC720 
AGCTTCGAGCCCATCCCCATCCACTACTGCGCCCCCGCCGGCTTCGCCATCCTGAAGTGC780 
AACGACAAGAAGTTCAGCGGCAAGGGCAGCTGCAAGAACGTGAGCACCGTGCAGTGCACC840 
CACGGCATCCGGCCGGTGGTGAGCACCCAGCTCCTGCTGAACGGCAGCCTGGCCGAGGAG900 
GAGGTGGTGATCCGCAGCGAGAACTTCACCGACAACGCCAAGACCATCATCGTGCACCTG960 
AATGAGAGCGTGCAGATCAACTGCACGCGTCCCAACTACAACAAGCGCAAGCGCATCCAC1020 
ATCGGCCCCGGGCGCGCCTTCTACACCACCAAGAACATCATCGGCACCATCCGCCAGGCC1080 
CACTGCAACATCTCTAGAGCCAAGTGGAACGACACCCTGCGCCAGATCGTGAGCAAGCTG1140 
AAGGAGCAGTTCAAGAACAAGACCATCGTGTTCAACCAGAGCAGCGGCGGCGACCCCGAG1200 
ATCGTGATGCACAGCTTCAACTGCGGCGGCGAATTCTTCTACTGCAACACCAGCCCCCTG1260 
TTCAACAGCACCTGGAACGGCAACAACACCTGGAACAACACCACCGGCAGCAACAACAAT1320 
ATTACCCTCCAGTGCAAGATCAAGCAGATCATCAACATGTGGCAGGAGGTGGGCAAGGCC1380 
ATGTACGCCCCCCCCATCGAGGGCCAGATCCGGTGCAGCAGCAACATCACCGGTCTGCTG1440 
CTGACCCGCGACGGCGGCAAGGACACCGACACCAACGACACCGAAATCTTCCGCCCCGGC1500 
GGCGGCGACATGCGCGACAACTGGAGATCTGAGCTGTACAAGTACAAGGTGGTGACGATC1560 
GAGCCCCTGGGCGTGGCCCCCACCAAGGCCAAGCGCCGCGTGGTGCAGCGCGAGAAGCGC1620 
TAAAGCGGCCGC1632 
(2) INFORMATION FOR SEQ ID NO:35: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2481 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:35: 
ACCGAGAAGCTGTGGGTGACCGTGTACTACGGCGTGCCCGTGTGGAAGGAGGCCACCACC60 
ACCCTGTTCTGCGCCAGCGACGCCAAGGCGTACGACACCGAGGTGCACAACGTGTGGGCC120 
ACCCAGGCGTGCGTGCCCACCGACCCCAACCCCCAGGAGGTGGAGCTCGTGAACGTGACC180 
GAGAACTTCAACATGTGGAAGAACAACATGCTGGAGCAGATGCATGAGGACATCATCAGC240 
CTGTGGGACCAGAGCCTGAAGCCCTGCGTGAAGCTGACCCCCCTGTGCGTGACCCTGAAC300 
TGCACCGACCTGAGGAACACCACCAACACCAACAACAGCACCGCCAACAACAACAGCAAC360 
AGCGAGGGCACCATCAAGGGCGGCGAGATGAAGAACTGCAGCTTCAACATCACCACCAGC420 
ATCCGCGACAAGATGCAGAAGGAGTACGCCCTGCTGTACAAGCTGGATATCGTGAGCATC480 
CACAACGACAGCACCAGCTACCGCCTGATCTCCTGCAACACCAGCGTGATCACCCAGGCC540 
TGCCCCAAGATCAGCTTCGAGCCCATCCCCATCCACTACTGCGCCCCCGCCGGCTTCGCC600 
ATCCTGAAGTGCAACGACAAGAAGTTCAGCGGCAAGGGCAGCTGCAAGAACGTGACCACC660 
GTGCAGTGCACCCACGGCATCCGGCCGGTGGTGAGCACCCAGCTCCTGCTGAACGGCAGC720 
CTGGCCGAGGAGGAGGTGGTGATCCGCAGCGAGAACTTCACCGACAACGCCAAGACCATC780 
ATCGTGCACCTGAATGAGAGCGTGCAGATCAACTGCACGCGTCCCAACTACAACAAGCGC840 
AAGCGCATCCACATCGGCCCCGGGCGCGCCTTCTACACCACCAAGAACATCATCGGCACC900 
ATCCGCCAGGCCCACTGCAACATCTCTAGAGCCAAGTGGAACGACACCCTGCGCCAGATC960 
GTGAGCAAGCTGAAGGAGCAGTTCAAGAACAAGACCATCGTGTTCAACCAGAGCAGCGGC1020 
GGCGACCCCGAGATCGTGATGCACAGCTTCAACTGCGGCGGCGAATTCTTCTACTGCAAC1080 
ACCAGCCCCCTGTTCAACAGCACCTGGAACGGCAACAACACCTGGAACAACACCACCGGC1140 
AGCAACAACAATATTACCCTCCAGTGCAAGATCAAGCAGATCATCAACATGTGGCAGGAG1200 
GTGGGCAAGGCCATGTACGCCCCCCCCATCGAGGGCCAGATCCGGTGCAGCAGCAACATC1260 
ACCGGTCTGCTGCTGACCCGCGACGGCGGCAAGGACACCGACACCAACGACACCGAAATC1320 
TTCCGCCCCGGCGGCGGCGACATGCGCGACAACTGGAGATCTGAGCTGTACAAGTACAAG1380 
GTGGTGACGATCGAGCCCCTGGGCGTGGCCCCCACCAAGGCCAAGCGCCGCGTGGTGCAG1440 
CGCGAGAAGCGGGCCGCCATCGGCGCCCTGTTCCTGGGCTTCCTGGGGGCGGCGGGCAGC1500 
ACCATGGGGGCCGCCAGCGTGACCCTGACCGTGCAGGCCCGCCTGCTCCTGAGCGGCATC1560 
GTGCAGCAGCAGAACAACCTCCTCCGCGCCATCGAGGCCCAGCAGCATATGCTCCAGCTC1620 
ACCGTGTGGGGCATCAAGCAGCTCCAGGCCCGCGTGCTGGCCGTGGAGCGCTACCTGAAG1680 
GACCAGCAGCTCCTGGGCTTCTGGGGCTGCTCCGGCAAGCTGATCTGCACCACCACGGTA1740 
CCCTGGAACGCCTCCTGGAGCAACAAGAGCCTGGACGACATCTGGAACAACATGACCTGG1800 
ATGCAGTGGGAGCGCGAGATCGATAACTACACCAGCCTGATCTACAGCCTGCTGGAGAAG1860 
AGCCAGACCCAGCAGGAGAAGAACGAGCAGGAGCTGCTGGAGCTGGACAACTGGGCGAGC1920 
CTGTGGAACTGGTTCGACATCACCAACTGGCTGTGGTACATCAAAATCTTCATCATGATT1980 
GTGGGCGGCCTGGTGGGCCTCCGCATCGTGTTCGCCGTGCTGAGCATCGTGAACCGCGTG2040 
CGCCAGGGCTACAGCCCCCTGAGCCTCCAGACCCGGCCCCCCGTGCCGCGCGGGCCCGAC2100 
CGCCCCGAGGGCATCGAGGAGGAGGGCGGCGAGCGCGACCGCGACACCAGCGGCAGGCTC2160 
GTGCACGGCTTCCTGGCGATCATCTGGGTCGACCTCCGCAGCCTGTTCCTGTTCAGCTAC2220 
CACCACCGCGACCTGCTGCTGATCGCCGCCCGCATCGTGGAACTCCTAGGCCGCCGCGGC2280 
TGGGAGGTGCTGAAGTACTGGTGGAACCTCCTCCAGTATTGGAGCCAGGAGCTGAAGTCC2340 
AGCGCCGTGAGCCTGCTGAACGCCACCGCCATCGCCGTGGCCGAGGGCACCGACCGCGTG2400 
ATCGAGGTGCTCCAGAGGGCCGGGAGGGCGATCCTGCACATCCCCACCCGCATCCGCCAG2460 
GGGCTCGAGAGGGCGCTGCTG2481 
(2) INFORMATION FOR SEQ ID NO:36: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 486 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:36: 
ATGAATCCAGTAATAAGTATAACATTATTATTAAGTGTATTACAAATGAGTAGAGGACAA60 
AGAGTAATAAGTTTAACAGCATGTTTAGTAAATCAAAATTTGAGATTAGATTGTAGACAT120 
GAAAATAATACACCTTTGCCAATACAACATGAATTTTCATTAACGCGTGAAAAAAAAAAA180 
CATGTATTAAGTGGAACATTAGGAGTACCAGAACATACATATAGAAGTAGAGTAAATTTG240 
TTTAGTGATAGATTCATAAAAGTATTAACATTAGCAAATTTTACAACAAAAGATGAAGGA300 
GATTATATGTGTGAGCTCAGAGTAAGTGGACAAAATCCAACAAGTAGTAATAAAACAATA360 
AATGTAATAAGAGATAAATTAGTAAAATGTGGAGGAATAAGTTTATTAGTACAAAATACA420 
AGTTGGTTATTATTATTATTATTAAGTTTAAGTTTTTTACAAGCAACAGATTTTATAAGT480 
TTATGA486 
(2) INFORMATION FOR SEQ ID NO:37: 
(i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 485 base pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: linear 
(xi) SEQUENCE DESCRIPTION: SEQ ID NO:37: 
ATGAACCCAGTCATCAGCATCACTCTCCTGCTTTCAGTCTTGCAGATGTCCCGAGGACAG60 
AGGGTGATCAGCCTGACAGCCTGCCTGGTGAACAGAACCTTCGACTGGACTGCCGTCATG120 
AGAATAACACCAACTTGCCCATCCAGCATGAGTTCAGCCTGACCCGAGAGAAGAAGAAGC180 
ACGTGCTGTCAGGCACCCTGGGGGTTCCCGAGCACACTTACCGCTCCCGCGTCAACCTTT240 
TCAGTGACCGCTTTATCAAGGTCCTTACTCTAGCCAACTTGACCACCAAGGATGAGGGCG300 
ACTACATGTGTGAACTTCGAGTCTCGGGCCAGAATCCCACAAGCTCCAATAAAACTATCA360 
ATGTGATCAGAGACAAGCTGGTCAAGTGTGGTGGCATAAGCCTGCTGGTTCAAAACACTT420 
CCTGGCTGCTGCTGCTCCTGCTTTCCCTCTCCTTCCTCCAAGCCACGGACTTCATTTCTC480 
TGTGA485 
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