E. coli msDNA synthesizing system, products and uses

A multicopy single-stranded DNA (msDNA) synthesizing system in E. coli is disclosed. The use of the msDNA system to synthesize cDNA in vivo is disclosed. Construction of synthetic msDNA is also disclosed. Also processes for gene amplification and for producing a stable RNA are disclosed.

The parent application discloses the presence of msDNA in the clinical E. 
coli isolate, C1-1, the cloned retron from the same strain and its 
nucleotide sequence. The instant continuation-in-part application 
additionally discloses a retron from another E. coli clinical isolate, 
C1-23, a process for in vivo cDNA production in E. coli and synthetic 
msDNA molecules. Additional disclosures of processes for gene 
amplification and production of stable RNA are also made. Large production 
of proteins is made possible by the invention. 
FIELD OF THE INVENTION 
This invention relates to a prokaryotic msDNA (multicopy single-stranded 
DNA) synthesizing system, also known as the retron. The invention also 
relates to msDNAs and to their production and their use to synthesize 
cDNA. The invention further relates to the use of one or more retron 
components in the production of various msDNAs. 
BACKGROUND OF THE INVENTION 
A novel satellite DNA called msDNA (multicopy single-stranded DNA) was 
originally found in Myxococcus xanthus, a Gram-negative bacterium living 
in soil (1). It consists of a 162-base single-stranded DNA, the 5' end of 
which is linked to a branched RNA (msdRNA) of 77 bases by a 
2',5'-phosphodiester linkage at the 2' position of the 20th rG residue 
(2). There are approximately 700 copies of msDNA per genome. msDNA is 
widely distributed among various myxobacteria including the closely 
related Stigmatella aurantiaca which possesses an msDNA, msDNA-Sa163. This 
molecule is highly homologous to msDNA-Mx162 from M. xanthus (3, 4). It is 
noteworthy that several M. xanthus strains, independently isolated from 
different sites, all contain msDNA (5). Recently it was found that M. 
xanthus contains another smaller species of msDNA called msDNA-Mx65 (6). 
In contrast to the close homology between msDNA-Mx162 and msDNA-Sa163, 
there is no primary sequence homology between msDNA-Mx162 and the small 
molecule, msDNA-Mx65. However, it was found that msDNA-Mx65 does share key 
secondary structures such as a branched rG residue, a DNA-RNA hybrid at 
the 3' ends of the msDNA and msdRNA, and stem-loop structures in RNA and 
DNA strands. 
It has been further shown that msdRNA is derived from a much longer 
precursor RNA (pre-msdRNA), which can form a very stable stem-and-loop 
structure (2). A novel mechanism for msDNA synthesis was proposed, in 
which the stem-and-loop structure of pre-msdRNA serves as a primer for 
initiating msDNA synthesis as well as a template to form the branched 
RNA-linked msDNA, and predicted that a reverse transcriptase (RT) is 
required for this reaction (2). 
Initial studies indicated that msDNA is not found in the common E. coli 
K-12 laboratory strain (1). To date, it has been observed that 
approximately 6% of all E. coli isolates from clinical strains carry an 
msDNA synthesizing system. This synthesizing system has been classified as 
a retron on the basis of rather surprising similarities between the msDNA 
and retroviruses and retrotransposons (8). 
The present invention provides for an E. coli msDNA synthesizing system. 
The invention also provides for its products and uses. 
BACKGROUND ART 
Bacterial reverse transcriptase and msDNA were initially discovered in 
Myxococcus xanthus and another myxobacterium Stigmatella aurantiaca. The 
publications noted here report on the myxobacteria discoveries. All such 
references are hereby incorporated by reference. 
Yee, T. and Inouye M. "Reexamination of the Genome Size of Myxobacteria, 
Including the Use of a New Method for Genome Size Analysis", J. Bacteriol. 
145, pp. 1257-1265 (1981), reports the discovery of a rapidly renaturing 
fraction of DNA found during the study of Myxobacteria genome size. 
Yee, T. et al., "Multicopy Single-Stranded DNA Isolated from a 
Gram-Negative Bacterium, Myxococcus xanthus", Cell 38, pp. 203-209 (1984), 
reports that the rapidly renaturing DNA found in Myxococcus xanthus is 
found as a satellite band upon polyacrylamide gel electrophoresis. This 
satellite DNA was called msDNA. Myxococcus xanthus was found to contain 
500 to 700 copies of msDNA per chromosome. The msDNA was cloned and 
sequenced. Its length and secondary structure was determined. A similar 
satellite DNA was found in the myxobacterium Stigmatella aurantiaca. The 
authors report that they were unable to detect any satellite DNA in 
Escherichia coli K-12. 
Furuichi, T. et al., "Branched RNA Covalently Linked to the 5' End of a 
Single-Stranded DNA in Stigmatella aurantiaca: Structure of msDNA", Cell 
48, pp. 47-53 (1987) and Furuichi, T. et al., "Biosynthesis and Structure 
of Stable Branched RNA Covalently Linked to the 5' End of Multicopy 
Single-Stranded DNA of Stigmatella aurantiaca", Cell 48, pp. 55-62 (1987), 
showed that msDNA isolated from S. aurantiaca (type Sa163) contained a DNA 
portion that was linked to an RNA molecule (msdRNA) by a 
2',5'-phosphodiester bond. The authors also reported that the coding 
region for msdRNA (msr) is located downstream of the coding region for 
msDNA (msd). The coding regions were found to exist in opposite 
orientation with respect to each other with their 3' ends overlapping. 
Dhundale, A. R. et al., "Distribution of Multicopy Single-Stranded DNA 
among Myxobacteria and Related Species", J. Bacteriol. 164, pp. 914-917 
(1985), examined how widely msDNA exists in various bacteria closely and 
distantly related to M. xanthus. msDNA was found in other myxobacteria and 
nine independently isolated strains of M. xanthus. The authors report 
msDNA to be found in certain gliding bacteria but not in others. 
The references cited above do not disclose or suggest that msDNA exists in 
E. coli. The publication of Yee et al. in Cell 38, 203 (1984) indicates 
that msDNA was undetectable in E. coli K-12 strain. The present invention 
encompasses recombinant DNA constructs encoding an E. coli msDNA 
synthesizing system and the components thereof. The unexpected discovery 
that about 6% of E. coli clinical isolates examined to date harbor msDNA 
enables the present invention. The present invention is thus a novel 
departure from the background art.

SUMMARY OF THE INVENTION 
Methods and compositions are provided for production of msDNA. The 
invention enables production of natural and synthetic msDNA. 
The invention provides for an msDNA synthesizing system. The three 
components of this system can be cloned in an E. coli expression vector as 
a unit or separately. The source of these components may be natural or 
synthetic. The components can also be utilized as they exist on the 
prokaryotic chromosome. 
The method of the invention provides for the utilization of the prokaryotic 
msDNA synthesizing system. The synthesizing system (retron) has three 
components, msd, msr and an ORF. Transcription and translation of the ORF 
region results in production of a protein having reverse transcriptase 
activity. Transcription of the msr region followed by DNA synthesis by 
reverse transcriptase results in msDNA production. 
The method of the invention provides for CDNA production within the cell. 
The invention provides an in vivo system to produce cDNA complementary to 
a specific RNA transcript in E. coli. Upon insertion of a sequence 
complementary to the 3' end of a msDNA molecule into a specific mRNA, CDNA 
to the mRNA is produced in vivo using msDNA as a primer. It is 
contemplated that CDNA could also be produced in vitro by providing an 
appropriate RNA, msDNA and reverse transcriptase. 
The invention also contemplates additional uses of artificial retrons as 
tools in life sciences research. Artificial msDNAs are contemplated to be 
useful for gene amplification, mRNA stabilization and production of 
ribozymes and antisense RNAs. Additionally, owing to ease of detection of 
msDNA, it is contemplated that bacteria producing msDNA can be used for 
the screening of antibodies and chemicals which block reverse 
transcriptase activity. 
DETAILED DESCRIPTION OF THE INVENTION 
This invention relates to a prokaryotic msDNA synthesizing system. This 
genetic system has been found in E. coli isolated from individuals with 
blood and urinary tract infections and those that are apparently healthy. 
The DNA fragment encoding the whole genetic system of msDNA has been 
classified as a retron since it appears to represent a primitive form of 
retroelement (8). It is proposed that the function of msDNA in the cell 
may be to serve as a primer to produce cDNA and the retron may function as 
a transposable element. FIG. 1 shows a restriction map of the retron. The 
DNA strand of the msDNA molecule is coded for by the msd gene. The RNA 
molecule of msDNA is encoded by the msr gene. The two genes are 
convergently situated (5' to 3') such that their respective 3' ends 
overlap. The third-retron component is an open reading frame (ORF) located 
upstream of msd and downstream of msr encoding a protein with reverse 
transcriptase activity (9). It is proposed that other ORFs may exist 
within the retron. These other ORFs may share sequence similarities with 
retroviral proteins such as integrase, protease and gag proteins. 
A population of E. coli clinical strains carry msDNA-synthesizing systems. 
At present, retrons have been found in approximately 6% (7 out of 113) of 
the clinical strains analyzed. It is contemplated that retrons exhibiting 
structural similarities exist in other genera of the Enterobacteriaceae 
family. 
Retrons from two E. coli clinical strains have been sequenced. The RNA and 
DNA sequence of the msDNAs produced by these retrons has also been 
determined. The complete primary and proposed secondary structure of these 
molecules (Ec67 and 74) are shown in FIG. 2. The numeric designation 
indicates the length of the DNA molecule. Little sequence homology is 
observed in both the RNA and DNA components of these molecules. However, 
despite their primary sequence differences, E. coli msDNAs all share key 
functional common features which include a single-stranded DNA with a 
stem-and-loop structure, a single-stranded RNA with a stem-and-loop 
structure, a 2',5'-phosphodiester linkage between RNA and DNA, and a 
DNA-RNA hybrid at the 3' ends. 
The invention also relates to the use of retron components in the 
production of various msDNAs and reverse transcriptases. The retron of the 
invention can be natural or synthetic. Two entire msr-msd regions have 
been synthesized using synthetic oligonucleotides and an example is 
illustrated in FIGS. 3 and 4. The region was inserted into a pINIII vector 
(14) (as a form of double-stranded DNA) such that a synthetic pre-msdRNA 
was produced in response to the addition of a lac inducer. The total gene 
length of approximately 200-bp was constructed by four units of 
double-stranded oligonucleotides. The gene was inserted into the unique 
XbaI site of the vector. The RT gene was provided in cis by inserting it 
into the same plasmid or in trans by inserting it in a separate plasnmid. 
It is thought that the retron can be utilized on the chromosome or 
extrachromosomally. It is contemplated that a naturally occurring retron 
can be altered through genetic engineering techniques. 
It is proposed that various artificial msDNAs can be constructed within the 
limitation of the requirements stated above. It is further proposed that 
the 3' end of msDNA can be variable, this part of the sequence can be 
substituted with a complementary sequence to a specific mRNA. Such an 
msDNA may be able to serve as a primer for the production of cDNA for a 
specific mRNA. 
msDNA-Ec67 retron is able to synthesize cDNA if cells contain an mRNA which 
has a stretch of RNA sequence complementary to the 3' end of msDNA-Ec67. A 
plasmid was constructed from pUC19 (13) which was able to produce an mRNA 
containing a sequence complementary to the 5' end of msDNA-Ec67 (FIG. 8). 
The sequence contained the 15-base sequence identical to the 3' end of 
msDNA-Ec67 such that the RNA transcript from pUC19 contains the 15-base 
sequence complementary to the 3' end of msDNA-Ec67 at position 80. 
When E. coli harboring the Ec67 retron capable of synthesizing msDNA-Ec67 
is transformed with plasmid pUC19-Ec67-20mer, the 3'-end region of 
Ec67-msDNA forms DNA-RNA hybrids not only with the 3' end of msdRNA (as 
shown in FIG. 2) but also with the RNA transcript from pUC19-Ec67-20-mer 
as shown in FIG. 9. Since the cells contain Ec67-RT, this enzyme starts to 
synthesize cDNA by extending the 3' end of msDNA along the mRNA template. 
A single-stranded DNA is synthesized of 152 bases which consists of the 
67-base msDNA at the 5' end and the 85-base cDNA to the 5' end of the lac 
transcript at the 3' end. Identification of this cDNA was made by 
polymerase chain reaction (PCR) (21). The results in FIG. 10A indicated a 
good agreement with the predicted 150-bp cDNA depicted in FIG. 9. An 
identical result was obtained with cells transformed with a pINIII vector 
(14) which also contained the same 20-bp sequence (FIG. 10, lanes 3 and 
5). 
The results described above are consistent with the cDNA structure depicted 
in FIG. 9, in which msDNA primes cDNA synthesis. To unambiguously prove 
this model, the DNA sequence of the PCR product with pUC19-Ec67-20-mer 
(FIG. 10A, lane 2) was determined. FIG. 11 shows the DNA sequence of the 
junction site, which clearly demonstrates that the 3' end of Ec67-msDNA is 
connected to the cDNA of the lac transcript of pUC19 at the 85th position. 
The present results unambiguously demonstrate that cDNA to a specific RNA 
transcript can be synthesized in E. coli cells by the method of the 
invention. This further indicates that cells are capable of producing cDNA 
if they contain RT and appropriate primers for a specific template. cDNA 
detected in the present study seems to exist mostly as single-stranded 
DNA, since cDNA production was detected by PCR after RNaseA treatment but 
not after treatment with S1 nuclease (See Example 5). Conversion of 
single-stranded cDNA to double-stranded cDNA may, however, easily occur in 
the cells if appropriate primers are provided. It is contemplated that an 
msDNA-synthesizing system could be established in eukaryotic cells. It is 
further contemplated that such a system may be used to obtain cDNA to a 
specific RNA transcript in vivo or cDNA to polyadenylated mRNAs in vivo by 
properly engineering the 3' ends of msDNA. 
It is also proposed that E. coli RT can synthesize cDNA from an mRNA if an 
appropriate primer is provided. An mRNA having a stable stem-and-loop 
structure at the 3' end may be able to prime cDNA synthesis by itself if 
an RT gene is expressed in the cell. 
Another contemplated approach is to use exogenously added synthetic 
oligonucleotide as primers which are complementary (antisense) to the 
mRNA. It is proposed that cells permeabilized with organic solvents will 
be useful for this method. 
The system of the invention is useful in various applications. Since msDNA 
is produced in several hundred copies per retron, the system can be used 
for gene amplification. This can be achieved by replacing the 
double-stranded region of msDNA with another double-stranded DNA 
containing a gene. In the synthetic msDNA depicted in FIG. 5, the 
stem-and-loop region (double-stranded region) of msDNA can be removed by 
restriction enzyme digestion of the retron-containing plasmid DNA with 
XhoI and SacII. A new DNA fragment is then ligated to this site, which 
contains two copies of a gene of interest either in head-to-head or in 
tail-to-tail orientation. As a result, when this region is copied as a 
single-stranded DNA in a synthetic msDNA, a secondary structure or a 
stem-and-loop structure is formed because of palindromic orientation of 
the two copies of the gene. Thus, the gene of interest is reconstructed in 
the stem structure. By this method of gene amplification, a large number 
of copies of the gene (e.g., more than 4,000), can be produced. This is 
provided that the plasmid containing msDNA sequences is maintained in E. 
coli at a copy number of 20 and that each plasmid produces 200 transcripts 
of the msDNA in a steady state. Of course, since the msDNA structure is 
not foreign to E. coli, the microorganism is particularly well suited as 
the vehicle for gene multiplication. 
In another application, the msDNA of the system of the invention are used 
to produce stable RNA. A DNA fragment can be inserted in the XbaI site 
located in the RNA structure (see FIG. 5). When the resulting retron is 
transcribed, RNA from the inserted DNA is added in the XbaI site of 
msdRNA. When the inserted DNA contains an open-reading frame, then the 
newly formed msdRNA functions as an mRNA containing the open-reading frame 
to produce a polypeptide. If the same DNA fragment is inserted in the 
opposite orientation, the newly synthesized msdRNA contains an RNA 
sequence complementary to the mRNA. Thus, it works as the antisense RNA 
against the mRNA, so that it can be used to regulate the expression of the 
gene for the mRNA. The RNA produced contains a Shine-Dalgarno sequence, an 
initiation codon and the coding sequence. This mRNA is extremely stable 
because of the 3' DNA-RNA hybrid structure, the secondary structure of the 
mRNA at the 3' end, and the branched rG residue at the 5' end. All these 
structures are considered to protect the mRNA from degradation. 
Thus, the invention provides a very useful system whereby a large amount of 
a specific mRNA is produced in a cell, resulting in expression of a large 
quantity of a specific polypeptide from the cloned gene. The industrial 
applications of the system are evident. High volume of a desired peptide 
can be comparatively inexpensively produced from the corresponding 
selected gene. Numerous valuable polypeptides can be produced like 
interferon, erythropoietin, plasminogen activators, antiplatelet 
aggregants, interlukin, growth and other hormones; and other biologically 
useful proteins. 
Another important application of the invention is that the msDNA be used to 
construct ribozymes or antisense RNAs or their combination. A DNA fragment 
can be inserted in the XbaI site so that the msdRNA synthesized from this 
construct contains a so-called hammer-head structure which works as a 
ribozyme, i.e., a ribonuclease which cleaves a specific RNA. Such a 
ribozyme can be used to destroy a specific mRNA. The XbaI site shown in 
FIG. 3 (see also FIG. 5) can be utilized for this purpose. If a hammerhead 
structure from a plant viroid (15-17) can be formed in the msdRNA at this 
site, a ribozyme is formed in msdRNA, which functions as a sequence 
specific ribonuclease. Similarly, if an antisense RNA against a specific 
gene is inserted at this site, the msDNA-antisense RNA may be very 
effective in blocking the expression of a specific gene. It is thought 
that a better suppression effect may occur upon combination of both 
ribozyme and antisense RNA within a single msdRNA. This approach leads to 
a new method for constructing more effective antisense RNA. The ribozyme 
functions to cleave selected other RNA molecules, e.g, specific viral 
RNAs. This antiviral approach can be usefully applied to a ribozyme 
specific as anti-HIV agent. The practical applications are evident in this 
area. 
It is also proposed that E. coli producing msDNA can be used for the 
screening of antibodies and chemicals which block RT activity. It is 
thought that anti-RT compounds will show stronger inhibitory effects on 
msDNA synthesis than on chromosomal DNA synthesis. 
The following examples illustrate the detection of msDNA in E. coli, 
nucleotide sequencing of msDNA and cloning of the msDNA genetic locus. The 
msDNAs and reverse transcriptases described herein are not limited to 
those specifically described herein. It can readily be seen by those 
skilled in the art that various msDNA molecules can be produced through 
synthetic means or genetic engineering. 
The following examples are only given for purposes of illustration and not 
by way of limitation on the, scope of the invention. 
EXAMPLE 1 
Detection of msDNA In E. coli 
Fifty independent E. coli urinary tract isolates identified with the use of 
the API-20E identification system (9) were examined for the presence of 
msDNA. Since msDNA contains a DNA-RNA-duplex structure, the 3' end of the 
DNA molecule serves as an intramolecular primer and the RNA molecule as a 
template for RT. When RNA prepared from one of the clinical strains, E. 
coli C1-1, was labeled in this manner, two distinct, low molecular weight 
bands of about 160 bases became labeled with .sup.32 P and are shown in 
FIG. 10. If the labeled sample is digested with ribonuclease (RNase) A 
prior to loading on the gel, a single band corresponding to 105 bases of 
single-stranded DNA is detected (lane 4). This indicates that both bands 
in lane 3 contain a single-stranded DNA of identical size. The two labeled 
bands observed prior to RNase treatment (lane 3) are due to two species of 
msDNA comprised of a single species of single-stranded DNA linked to RNA 
molecules of two different sizes. Among the fifty clinical isolates 
screened, three other strains produced msDNA-like molecules of varying 
size and quantity suggesting extensive diversity among these molecules. 
In a similar experiment, RNA was extracted from 113 independent clinical 
isolates. Fifty were from patients with a urinary tract infection, and 63 
from patients with blood infections. Among the 50 strains from patients 
with a urinary tract infection, three were found to contain msDNA. From 
patients with blood infections, 3 strains were found to contain msDNA. In 
addition, msDNAs have been found in E. coli strains from apparently normal 
human stool samples; msDNA was not observed in the E. coli K-12 strain, 
C600. 
EXAMPLE 2 
Nucleotide sequence of msDNA 
To determine the base sequence of the DNA molecule, the RNA-DNA complex 
isolated from the clinical stain was labeled at the 3' end of the DNA 
molecule with AMV-RT and [.alpha.-.sup.32 P]dATP. By adding ddCTP, ddTTP, 
and ddGTP to the reaction mixture, a single labeled adenine is added to 
the 3' end of the DNA molecule. RNA is removed with RNase A+T and the 
end-labeled DNA is subjected to the Maxam and Gilbert sequencing method 
(3). FIG. 2 shows that this msDNA consists of a single-stranded DNA of 67 
bases and that it can form a secondary hair-pin structure. Accordingly, 
this msDNA has been denoted as msDNA-Ec67. 
The sequence of the RNA molecules was determined using the RNA-DNA complex 
purified from E. coli C1-1 as described in Example 1. As shown in FIG. 11, 
a large gap is observed in the RNA sequence "ladder". This gap is due to 
the DNA strand branched at the 2' position of the 15th rG residue of the 
RNA strand which produces a shift in mobility of the sequence ladder (see 
FIG. 2). The RNA consists of 58 bases with the DNA molecule branched at 
the G residue at position 15 by a 2',5'-phosphodiester linkage. The 
branched G structure was determined as described for msDNAs from 
myxobacteria (5, 6). After RNase (A and T.sub.1) treatment, msDNA retains 
a small oligoribonucleotide linked to the 5' end of the DNA molecule due 
to the inability of RNases to cleave in the vicinity of the branched 
linkage. The 5' end was labeled with [.gamma.-.sup.32 P] ATP using T.sub.4 
polynucleotide kinase and the labeled RNA molecule was detached from the 
DNA strand by a debranching enzyme purified from HeLa cells (5, 6). This 
small RNA was found to be a tetraribonucleotide which could de digested 
with RNase T.sub.1 to yield a labeled dinucleotide. Since RNase T.sub.1 
could not cleave the RNA molecule at the G residue before debranching 
enzyme treatment, it was concluded that the single-stranded DNA is 
branched at the G residue via a 2',5'-phosphodiester linkage. In addition, 
partial RNase U.sub.2 digestion cleaved the RNA molecule to yield a 
.sup.32 P-labeled mono- and a .sup.32 P-labeled trinucleotide. Thus, the 
sequence of the tetranucleotide is .sup.5' A-G-A-(U or C).sup.3'. Based on 
these data, the complete structure of msDNA-Ec67 from E. coli C1-1 is 
presented in FIG. 2. Despite a lack of primary and structural homology, 
msDNA-Ec67 displays all the unique features found in msDNAs from 
myxobacteria. These include a single-stranded DNA with a stem-and-loop 
structure, a single-stranded RNA with a stem-and-loop structure, a 
2',5'-phosphodiester linkage between the RNA and DNA, and a DNA-RNA hybrid 
at their 3' ends. This hybrid structure was confirmed by demonstrating 
sensitivity of the RNA molecule to RNaseH. 
EXAMPLE 3 
Cloning of the locus for msDNA 
In order to identify the DNA fragment which is responsible for msDNA 
synthesis in E. coli C1-1, DNA blot hybridization (18) was carried out 
with various restriction enzyme digests of total chromosomal DNA prepared 
from E. coli C1-1, using msDNA-Ec67 labeled with AMV-RT (the same 
preparation as shown in lane 3, FIG. 10) as a probe. For each lane, 3 
.mu.g of the DNA digest was applied to a 0.7% agarose gel. The result is 
shown in FIG. 12A EcoRI (lane 1), HindIlI (lane 2), BamHI (lane 3), PstI 
(lane 4) and BglII (lane 5) digestions showed single band hybridization 
signals corresponding to 11.6, 2.0, approximately 22, 2.8 and 2.5 kilobase 
pairs (kb), respectively. The upper band appearing in the EcoRI digestion 
is due to incomplete digestion of the chromosomal DNA. Analysis of total 
chromosomal DNA prepared form E. coli C1-1 by agarose gel electrophoresis 
revealed that the strain contains two plasmids of different size. However, 
neither plasmid hybridized with the .sup.32 P-labeled probe, indicating 
the fragments detected in FIG. 12A are derived from chromosomal DNA. 
Furthermore, there is only one location for the msDNA-coding region on the 
chromosome, since various restriction enzyme digestions gave only one band 
of varying sizes. 
The 11.6-kb EcoRI fragment and the 2.8-kb PstI fragment were each cloned 
into pUC9 (9) and E. coli CL83 (a recA transductant of strain JM83), an 
msDNA-free K-12 strain (lane 1, FIG. 12B) was transformed with the 
plasmids. Cells transformed with the 11.6-kb EcoRI clone (pC1-1E) were 
found to produce msDNA (lane 2, FIG. 12B, whereas cells transformed with 
the 2.8-kb PstI clone (pC1-1P) failed to produce any detectable msDNA 
(lane 3, FIG. 12B). A map of the 11.6-kb fragment is shown in FIG. 1. DNA 
blot analysis of the fragment revealed that a 1.8-kb PstI-HindIII fragment 
hybridized with the msDNA probe. When the DNA sequence of this fragment 
was determined, a region identical to the sequence of the msDNA molecule 
was discovered. The DNA sequence corresponding to the sequence of msDNA is 
indicated by the enclosed box on the lower strand in FIG. 7 and the 
orientation is from right to left. The location of this sequence is also 
indicated by a small arrow in FIG. 1. A sequence identical to that of the 
RNA linked to msDNA (see FIG. 2) was found downstream of the msDNA-coding 
region in opposite orientation and overlapping with the region by 7 bases. 
This sequence is indicated by the enclosed box on the upper strand in FIG. 
13 and the branched G residue is circled. Again, as in all the msDNAs 
found in myxobacteria, there is an inverted repeat comprised of a 13-base 
sequence immediately upstream of the branched G residue (residue 250 to 
262; sequence a2 in FIG. 13) and a sequence at the 3' end shown by an 
arrow in FIG. 13 (residue 368 to 380; sequence a1). As a result of this 
inverted repeat, a putative longer primary RNA transcript beginning 
upstream of the RNA coding region and extending through the msDNA coding 
region would be able to self-anneal and form a stable secondary structure, 
which is proposed to serve as the primer as well as the template for the 
msDNA synthesis (5). 
EXAMPLE 4 
Construction of Synthetic msDNA 
Two distinct synthetic msDNA molecules were constructed. A 196-bp synthetic 
msDNA containing an entire msr-msd region was synthesized from four 
double-stranded oligonucleotide units. The synthetic genes and their 
components are shown in FIG. 3. Eight single-stranded oligonucleotides, 
forty-six to fifty-six bases in length were synthesized. The appropriate 
pairs of oligonucleotides were annealed by heating at 100.degree. C. for 5 
minutes, then cooling at 30.degree. C. for 30 minutes and for 30 minutes 
in a refrigerator. An E. coli pINIII(lpp.sup.p-5) expression vector (14) 
was digested with XbaI-EcoRI and an XbaI-EcoRI fragment from the clinical 
E. coli strain C1-1 was inserted such that the RT gene under lpp-lac 
promoter control and used to transform E. coli. After identification of 
the clone, the 10.7-kb pINIII(lpp.sup.p-5) Ec67-RT plasmid DNA was 
isolated. The 196-bp synthetic msDNA fragment was then inserted into the 
vector by digesting with XbaI, treating the vector ends with bacterial 
alkaline phosphatase and ligating the fragment into the site. The 
construction scheme is shown in FIG. 4. E. coli CL-83 was transformed with 
the pINIII(lpp.sup.p-5) ms100-RT plasmid and the production of msDNA 
determined as in Example 1. The results indicated that msDNA was produced. 
This artificial msDNA was designated ms100 and is illustrated in FIG. 5. 
A second synthetic msDNA, ms101, was expressed from the vector pUCK19, a 
derivative of pUC19 (13). pUC19 DNA was digested with DraI and the 2-kb 
fragment isolated. The isolated fragment was ligated to a 1.3-kb HinfI 
fragment from Tn5 encoding the kanamycin resistance gene. The resultant 
3.3-kb plasmid, pUCK19, was digested with XbaI and the 196-bp synthetic 
msDNA described above was inserted. The pUCKms100 construct was digested 
with XhoI and SacII which results in the excision of a 61-bp fragment from 
within the ms100 region. A synthetic 45-mer double-stranded 
oligonucleotide (shown in FIG. 3B as ms-C1,2) was ligated into the vector 
yielding pUCKms101 in which the msr-msd region is under lac control. The 
construction scheme is shown in FIG. 6. RT was provided by transforming E. 
coli containing pU CKms100 or pUCKms101 with pINIII (lpp.sup.p-5) Ec67-RT. 
msDNA production was detected in the cells containing these constructs. 
ms101 is shown in FIG. 7. 
EXAMPLE 5 
In Vivo CDNA Production in E. coli 
In order to test a cDNA production in E. coli, a plasmid was constructed 
which was able to produce an IRNA containing a sequence complementary to 
the 5' end of msDNA-Ec67. The construction of this plasmid 
(pUC19-Ec67-20), in which a 20-bp sequence was added at the unique XbaI 
site of pUC19 is illustrated in FIG. 9. The 20-bp sequence contains a 
15-base sequence identical to the 3' end of msDNA-Ec67 (see FIG. 2A) so 
that the RNA transcript from the lac promoter of pUC19 contains the 
15-base sequence complementary to the 3' end of msDNA-Ec67 at the position 
80 bases downstream of the 5' end of the transcript. 
If E. coli JA221 harboring the Ec67 retron (pC1-1EP5b), is transformed 20 
with plasmid pUC19-Ec67-20, the 3'-end region of msDNA-Ec67 may form a 
DNA-RNA hybrid not only with the 3' end of msdRNA (as shown in FIG. 2A) 
but also with the RNA transcript (lacZ mRNA) from pUC19-Ec67-20 as shown 
in FIG. 9. Since the cells contain Ec67-RT, this enzyme may then start to 
synthesize cDNA by extending the 3' end of msDNA along the mRNA template. 
This would produce a single-stranded DNA of 152 bases which consists of 
the 67-base msDNA at the 5' end and the 85-base cDNA (to the 5' end of the 
lac transcript; 80 bases from the lacZ mRNA plus 5 bases from the linker) 
at the 3' end. Identification of this cDNA was carried out by the 
polymerase chain reaction (PCR) (19) using a 23-base oligonucleotide 
complementary to the 3' end of the cDNA (P1; see FIG. 9) and a 23-base 
oligonucleotide identical to the 5' end of msDNA-Ec67 (P2) as primers. A 
DNA fraction containing cDNA was digested with ribonuclease A and then 
used for the PCR. After the 25th cycle of the PCR, the DNA products were 
fractionated on a 5% polyacrylamide gel and detected by staining with 
ethidium bromide. A distinct band appeared at the position of 
approximately 150-base pairs. This band yielded two bands of approximately 
80 and 70-base pairs after XbaI, PstI and HindIII digestion. This is in 
good agreement with the predicted 152-bp cDNA depicted in FIG. 9, which is 
expected to yield two fragments of 80 and 72-bp upon XbaI digestion. The 
PCR did not yield any specific bands when pUC19 without the 20-bp insert 
was used. 
FIG. 13 shows nucleotide sequence of the region from the E. coli C1-1 
chromosome encompassing the msDNA and the msdRNA coding regions and an ORF 
downstream of the msdRNA region. The entire upper beginning at the BalI 
site and ending just beyond the ORF is shown. Only a part of the 
complementary lower strand is shown from base 241. to 420. The long boxed 
region of the upper strand (249-306) corresponds to the sequence of the 
branched RNA portion of msDNA molecule. The boxed region of the lower 
strand corresponds to the sequence of the DNA portion of msDNA. The 
starting site for DNA and RNA and the 5' to 3' orientation are indicated 
by large open arrows. The msdRNA and msDNA regions overlap at their 3' 
ends by 7 bases. The circled G residues at position 263 represents the 
branched rG of RNA linked to the 5' end of the DNA strand in msDNA. Long 
solid arrows labeled a1 and a2 represent inverted repeat sequences 
proposed to be important in the secondary structure of the primary RNA 
transcripts involved in the synthesis of msDNA. Note that the nucleotide 
at position 257 (U on the RNA transcript) form an U-G pair in the stem 
between sequence a1 and a2. The proposed promoter elements (-10 and -35 
regions) for the primary RNA transcript are also boxed. The ORF coding for 
586 amino acid residues begins with the initiation codon ATG at base 
418-420 to end with nucleotide 2175. Single letter designations are given 
for amino acids. The YXDD amino acid sequence conserved among known RT 
proteins is boxed. Numbers on the right hand column enumerate the 
nucleotide bases and numbers with a * enumerate amino acids. Small 
vertical arrows labelled H and P located the HindIII and PstI restriction 
cleavage sites, respectively. The DNA sequence was determined by the chain 
termination method using synthetic oligonucleotides as primers. 
REFERENCES 
1. Yee, T. et al., Cell 38, 203 (1984). 
2. Dhundale, A. et al., Cell 51, 1105 (1987). 
3. Furuichi, T. et al., Cell 48, 47 (1987). 
4. Furuichi, T. et al., Cell 48, 55 (1987). 
5. Dhundale, A. et al., J. Bacteriol. 164, 914 (1985). 
6. Dhundale, A. et al., J. Biol. Chem. 263, 9055 (1988). 
7. Lim, D. and Maas, W., Cell 56, 891 (1989). 
8. Temin, H. M., Nature 339, 254 (1989). 
9. Lampson, B. C. et al., Science 243, 1033 (1989). 
10. Maxam, A. M. and Gilbert, W., Methods Enzymol. 65, 499 (1980). 
11. Ruskin, B. and Green, M., Science 229, 135 (1985). 
12. Arenas, J. and Hurwitz, J., J. Biol. Chem. 262, 4274 (1987). 
13. Yanisch-Perron, Y. et al., Gene 33, 103 (1985). 
14. Masui, Y. et al., "Experimental Manipulation of Gene Expression" (ed. 
M. Inouye), pp. 15-32, Academic Press, New York (1983). 
15. Hutchins, C. J. et al., Nucl. Acids Res. 14, 3627 (1986). 
16. Foster, A. C. and Symons, R. H., Cell 49, 211 (1987). 
17. Coleman, J. et al., Cell 37, 429 (1984). 
18. Southern, E., J. Mol. Biol. 98, 503 (1975). 
19. Saiki, R. K. et al., Science 230, 1350 (1985). 
__________________________________________________________________________ 
# SEQUENCE LISTING 
- - - - (1) GENERAL INFORMATION: 
- - (iii) NUMBER OF SEQUENCES: 12 
- - - - (2) INFORMATION FOR SEQ ID NO:1: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 58 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: both 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 
- - CACGCAUGUA GGCAGAUUUG UUGGUUGUGA AUCGCAACCA GUGGCCUUAA UG - 
#GCAGGA 58 
- - - - (2) INFORMATION FOR SEQ ID NO:2: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 67 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: both 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: 
- - TCCTTCGCAC AGCACACCTG CCGTATAGCT CTGAATCAAG GATTTTAGGG AG - 
#GCGATTCC 60 
- - TCCTGCC - # - # 
- # 67 
- - - - (2) INFORMATION FOR SEQ ID NO:3: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 70 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: both 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: 
- - CCAAACCUAG CAUUUUAUGG GUUAAUAGCC CAUCGCGCAU GAGUCAUGGU UU - 
#CGCCUAGU 60 
- - AUUUUAGCUA - # - # 
- # 70 
- - - - (2) INFORMATION FOR SEQ ID NO:4: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 74 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: both 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: 
- - TTGAGCACGT CGATCAGTTC GCTGATCGGT GGCCCCCCAG CCGCCGCTCA GC - 
#GAATTGAA 60 
- - CGACGGGCAT AGCT - # - # 
- # 74 
- - - - (2) INFORMATION FOR SEQ ID NO:5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 200 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5: 
- - CTAGTGATAT GTTCATAAAC ACGCATGTAG GCAGATTCTA GATTGGTTGT GA - 
#ATCGCAAC 60 
- - CAGTGGCCTT ATGGCAGGAG CCGCGGATCA CCTACCATCC CTAATATTCT CT - 
#TTCAGAGA 120 
- - ATATTAGGTA CGGCAGGTGT GCTCGAGGCG AAGGAGTGCC TGCATGCGTT TC - 
#TCCTTGGC 180 
- - CTTTTTCCTC TGGGAACTAG - # - # 
- #200 
- - - - (2) INFORMATION FOR SEQ ID NO:6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 
- - GCGGGCCTCC CTAAAATCCT TGATTCAGAG CTATACGGCA GGTGTGCTCG A - # 
51 
- - - - (2) INFORMATION FOR SEQ ID NO:7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 60 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: both 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: 
- - CACGCAUGUA GGCAGAUUCU AGAUUGGUUG UGAAUCGCAA CCAGUGGCCU UA - 
#UGGCAGGA 60 
- - - - (2) INFORMATION FOR SEQ ID NO:8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 83 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: both 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: 
- - TCCTTCGCCT CGAGCACACC TGCCGTACCT AATATTCTCT GAAAGAGAAT AT - 
#TAGGGATG 60 
- - GTAGGTGATC CGCGGCTCCT GCC - # - # 
83 
- - - - (2) INFORMATION FOR SEQ ID NO:9: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 60 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: both 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: 
- - CACGCAUGUA GGCAGAUUCU AGAUUGGUUG UGAAUCGCAA CCAGUGGCCU UA - 
#UGGCAGGA 60 
- - - - (2) INFORMATION FOR SEQ ID NO:10: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 70 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: single 
(D) TOPOLOGY: both 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: 
- - TCCTTCGCCT CGAGCACACC TGCCGTATAG CTCTGAATCA AGGATTTTAG GG - 
#AGGCCCGC 60 
- - GGCTCCTGCC - # - # 
- # 70 
- - - - (2) INFORMATION FOR SEQ ID NO:11: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 2423 base - #pairs 
(B) TYPE: nucleic acid 
(C) STRANDEDNESS: double 
(D) TOPOLOGY: linear 
- - (ix) FEATURE: 
(A) NAME/KEY: CDS 
(B) LOCATION: 418..2175 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: 
- - TGGCCATTCA GATACGGATT TTCACTTCCT TGACAGTGCA TGACTATGCT GC - 
#ATGAAATC 60 
- - GCATGATCGA TTGAGGATCG TCTTTGCTCA GATCCGCCAG AACTGGCGGG CT - 
#TTTGCTCA 120 
- - TGTCATGCAT GTGCATGAAA ACCACTGCAT AAAGCGGGCA GGCCTGGCGG GG - 
#ATACGAGC 180 
- - GCGCGCTATC ACCGAAAATA GCCAAAATAC TTCTGGAAAA CAGAAAGTTG AA - 
#CTGATATC 240 
- - TTCATAAACA CGCATGTAGC CAGATTTGTT GGTTGTGAAT CGCAACCAGT GG - 
#CCTTAATG 300 
- - GCAGGAGGAA TCGCCTCCCT AAAATCCTTG ATTCAGAGCT ATACGGCAGG TG - 
#TGCTGTGC 360 
- - GAAGGACTGC CTGCATGCGT TTCTCCTTGG CCTTTTTTCC TCTGGGATGA AG - #AAGAA 
417 
- - ATG ACA AAA ACA TCT AAA CTT GAC GCA CTT AG - #G GCT GCT ACT TCA CGT 
465 
Met Thr Lys Thr Ser Lys Leu Asp Ala Leu Ar - #g Ala Ala Thr Ser Arg 
1 5 - # 10 - # 15 
- - GAA GAC TTG GCT AAA ATT TTA GAT ATT AAG TT - #G GTA TTT TTA ACT AAC 
513 
Glu Asp Leu Ala Lys Ile Leu Asp Ile Lys Le - #u Val Phe Leu Thr Asn 
20 - # 25 - # 30 
- - GTT CTA TAT AGA ATC GGC TCG GAT AAT CAA TA - #C ACT CAA TTT ACA ATA 
561 
Val Leu Tyr Arg Ile Gly Ser Asp Asn Gln Ty - #r Thr Gln Phe Thr Ile 
35 - # 40 - # 45 
- - CCG AAG AAA GGA AAA GGG GTA AGG ACT ATT TC - #T GCA CCT ACA GAC CGG 
609 
Pro Lys Lys Gly Lys Gly Val Arg Thr Ile Se - #r Ala Pro Thr Asp Arg 
50 - # 55 - # 60 
- - TTG AAG GAC ATC CAA CGA AGA ATA TGT GAC TT - #A CTT TCT GAT TCT AGA 
657 
Leu Lys Asp Ile Gln Arg Arg Ile Cys Asp Le - #u Leu Ser Asp Ser Arg 
65 - # 70 - # 75 - # 80 
- - GAT GAG ATC TTT GCT ATA AGG AAA ATT AGT AA - #C AAC TAT TCC TTT GCT 
705 
Asp Glu Ile Phe Ala Ile Arg Lys Ile Ser As - #n Asn Tyr Ser Phe Ala 
85 - # 90 - # 95 
- - TTT GAG AGG GGA AAA TCA ATA ATC CTA AAT GC - #T TAT AAG CAT AGA GGC 
753 
Phe Glu Arg Gly Lys Ser Ile Ile Leu Asn Al - #a Tyr Lys His Arg Gly 
100 - # 105 - # 110 
- - AAA CAA ATA ATA TTA AAT ATA GAT CTT AAG GA - #T TTT TTT GAA AGC TTT 
801 
Lys Gln Ile Ile Leu Asn Ile Asp Leu Lys As - #p Phe Phe Glu Ser Phe 
115 - # 120 - # 125 
- - AAT TTT GGA CGA GTT AGA GGA TAT TTT CTT TC - #C AAT CAG GAT TTT TTA 
849 
Asn Phe Gly Arg Val Arg Gly Tyr Phe Leu Se - #r Asn Gln Asp Phe Leu 
130 - # 135 - # 140 
- - TTA AAT CCT GTG GTG GCA ACG ACA CTT GCA AA - #A GCT GCA TGC TAT AAT 
897 
Leu Asn Pro Val Val Ala Thr Thr Leu Ala Ly - #s Ala Ala Cys Tyr Asn 
145 1 - #50 1 - #55 1 - 
#60 
- - GGA ACC CTC CCC CAA GGA AGT CCA TGT TCT CC - #T ATT ATC TCA AAT 
CTA 945 
Gly Thr Leu Pro Gln Gly Ser Pro Cys Ser Pr - #o Ile Ile Ser Asn Leu 
165 - # 170 - # 175 
- - ATT TGC AAT ATT ATG GAT ATG AGA TTA GCT AA - #G CTG GCT AAA AAA TAT 
993 
Ile Cys Asn Ile Met Asp Met Arg Leu Ala Ly - #s Leu Ala Lys Lys Tyr 
180 - # 185 - # 190 
- - GGA TGT ACT TAT AGC AGA TAT GCT GAT GAT AT - #A ACA ATT TCT ACA AAT 
1041 
Gly Cys Thr Tyr Ser Arg Tyr Ala Asp Asp Il - #e Thr Ile Ser Thr Asn 
195 - # 200 - # 205 
- - AAA AAT ACA TTT CCG TTA GAA ATG GCT ACT GT - #G CAA CCT GAA GGG GTT 
1089 
Lys Asn Thr Phe Pro Leu Glu Met Ala Thr Va - #l Gln Pro Glu Gly Val 
210 - # 215 - # 220 
- - GTT TTG GGA AAA GTT TTG GTA AAA GAA ATA GA - #A AAC TCT GGA TTC GAA 
1137 
Val Leu Gly Lys Val Leu Val Lys Glu Ile Gl - #u Asn Ser Gly Phe Glu 
225 2 - #30 2 - #35 2 - 
#40 
- - ATA AAT GAT TCA AAG ACT AGG CTT ACG TAT AA - #G ACA TCA AGG CAA 
GAA 1185 
Ile Asn Asp Ser Lys Thr Arg Leu Thr Tyr Ly - #s Thr Ser Arg Gln Glu 
245 - # 250 - # 255 
- - GTA ACG GGA CTT ACA GTT AAC AGA ATC GTT AA - #T ATT GAT AGA TGT TAT 
1233 
Val Thr Gly Leu Thr Val Asn Arg Ile Val As - #n Ile Asp Arg Cys Tyr 
260 - # 265 - # 270 
- - TAT AAA AAA ACT CGG GCG TTG GCA CAT GCT TT - #G TAT CGT ACA GGT GAA 
1281 
Tyr Lys Lys Thr Arg Ala Leu Ala His Ala Le - #u Tyr Arg Thr Gly Glu 
275 - # 280 - # 285 
- - TAT AAA GTG CCA GAT GAA AAT GGC GTT TTA GT - #T TCA GGA GGT CTG GAT 
1329 
Tyr Lys Val Pro Asp Glu Asn Gly Val Leu Va - #l Ser Gly Gly Leu Asp 
290 - # 295 - # 300 
- - AAA CTT GAG GGG ATG TTT GGT TTT ATT GAT CA - #A GTT GAT AAG TTT AAC 
1377 
Lys Leu Glu Gly Met Phe Gly Phe Ile Asp Gl - #n Val Asp Lys Phe Asn 
305 3 - #10 3 - #15 3 - 
#20 
- - AAT ATA AAG AAA AAA CTG AAC AAG CAA CCT GA - #T AGA TAT CTA TTG 
ACT 1425 
Asn Ile Lys Lys Lys Leu Asn Lys Gln Pro As - #p Arg Tyr Leu Leu Thr 
325 - # 330 - # 335 
- - AAT GCG ACT TTG CAT GGT TTT AAA TTA AAG TT - #G AAT GCG CGA GAA AAA 
1473 
Asn Ala Thr Leu His Gly Phe Lys Leu Lys Le - #u Asn Ala Arg Glu Lys 
340 - # 345 - # 350 
- - GCA TAT ACT AAA TTT ATT TAC TAT AAA TTT TT - #T CAT GGC AAC ACC TGT 
1521 
Ala Tyr Thr Lys Phe Ile Tyr Tyr Lys Phe Ph - #e His Gly Asn Thr Cys 
355 - # 360 - # 365 
- - CCT ACG ATA ATT ACA GAA GGG AAG ACT GAT CG - #G ATA TAT TTG AAG GCT 
1569 
Pro Thr Ile Ile Thr Glu Gly Lys Thr Asp Ar - #g Ile Tyr Leu Lys Ala 
370 - # 375 - # 380 
- - GCT TTG CAT TCT TTG GAG ACA TCA TAT CCT GA - #G TTG TTT AGA GAA AAA 
1617 
Ala Leu His Ser Leu Glu Thr Ser Tyr Pro Gl - #u Leu Phe Arg Glu Lys 
385 3 - #90 3 - #95 4 - 
#00 
- - ACA GAT AGT AAA AAG AAA GAA ATA AAT CTT AA - #T ATA TTT AAA TCT 
AAT 1665 
Thr Asp Ser Lys Lys Lys Glu Ile Asn Leu As - #n Ile Phe Lys Ser Asn 
405 - # 410 - # 415 
- - GAA AAG ACC AAA TAT TTT TTA GAT CTT TCT GG - #G GGA ACT GCA GAT CTG 
1713 
Glu Lys Thr Lys Tyr Phe Leu Asp Leu Ser Gl - #y Gly Thr Ala Asp Leu 
420 - # 425 - # 430 
- - AAA AAA TTT CTA GAG CGT TAT AAA AAT AAT TA - #T GCT TCT TAT TAT GCT 
1761 
Lys Lys Phe Leu Glu Arg Tyr Lys Asn Asn Ty - #r Ala Ser Tyr Tyr Ala 
435 - # 440 - # 445 
- - TCT GTT CCA AAA CAC CCA GTG ATT ATG CTT CT - #T GAT AAT GAT ACA GCT 
1809 
Ser Val Pro Lys His Pro Val Ile Met Leu Le - #u Asp Asn Asp Thr Ala 
450 - # 455 - # 460 
- - CCA AGC GAT TTA CTT AAT TTT CTG CGC AAT AA - #A GTT AAA AGC TGC CCA 
1857 
Pro Ser Asp Leu Leu Asn Phe Leu Arg Asn Ly - #s Val Lys Ser Cys Pro 
465 4 - #70 4 - #75 4 - 
#80 
- - GAC GAT GTA ACT GAA ATG AGA AAG ATG AAA TA - #T ATT CAT CTT TTC 
TAT 1905 
Asp Asp Val Thr Glu Met Arg Lys Met Lys Ty - #r Ile His Leu Phe Tyr 
485 - # 490 - # 495 
- - AAT TTA TAT ATA CTT CTC ACA CCA TTG AGT CC - #T TCC GGC GAA CAA ACT 
1953 
Asn Leu Tyr Ile Leu Leu Thr Pro Leu Ser Pr - #o Ser Gly Glu Gln Thr 
500 - # 505 - # 510 
- - TCA ATG GAG GAT CTT TTC CCT AAA GAT ATT TT - #A GAT ATC AAG ATT GAT 
2001 
Ser Met Glu Asp Leu Phe Pro Lys Asp Ile Le - #u Asp Ile Lys Ile Asp 
515 - # 520 - # 525 
- - GGT AAG AAA TTC AAC AAA AAT AAT GAT GGA GA - #C TCA AAA ACG GAA TAT 
2049 
Gly Lys Lys Phe Asn Lys Asn Asn Asp Gly As - #p Ser Lys Thr Glu Tyr 
530 - # 535 - # 540 
- - GGG AAG CAT ATT TTT TCC ATG AGG GTT GTT AG - #A GAT AAA AAG CGG AAA 
2097 
Gly Lys His Ile Phe Ser Met Arg Val Val Ar - #g Asp Lys Lys Arg Lys 
545 5 - #50 5 - #55 5 - 
#60 
- - ATA GAT TTT AAG GCA TTT TGT TGT ATT TTT GA - #T GCT ATA AAA GAT 
ATA 2145 
Ile Asp Phe Lys Ala Phe Cys Cys Ile Phe As - #p Ala Ile Lys Asp Ile 
565 - # 570 - # 575 
- - AAG GAA CAT TAT AAA TTA ATG TTA AAT AGC TA - #ATGAACAG CCCTAACGTT 
2195 
Lys Glu His Tyr Lys Leu Met Leu Asn Ser 
580 - # 585 
- - ATGAACGCTA AGGCTGATTT TTCGTTAAAA TTTATATGCT TTGAATTGTA AT - 
#ATATTATC 2255 
- - TTCAAGCCAT TTATTTAATT CCTGCATCCT TTTCTCTAAG GGTATTAATT CG - 
#TTCCTCAC 2315 
- - AAACACTAAA CTCGCTTTTT CCACATCCCC AAACCCCCCT AACATTATTC GG - 
#CATAATCC 2375 
- - CCATCATTTG CGGTGGCACA CCATGCGCTG CCATCATCTC ATCGCGGC - # 
2423 
- - - - (2) INFORMATION FOR SEQ ID NO:12: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 586 amino - #acids 
(B) TYPE: amino acid 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: 
- - Met Thr Lys Thr Ser Lys Leu Asp Ala Leu Ar - #g Ala Ala Thr Ser Arg 
1 5 - # 10 - # 15 
- - Glu Asp Leu Ala Lys Ile Leu Asp Ile Lys Le - #u Val Phe Leu Thr Asn 
20 - # 25 - # 30 
- - Val Leu Tyr Arg Ile Gly Ser Asp Asn Gln Ty - #r Thr Gln Phe Thr Ile 
35 - # 40 - # 45 
- - Pro Lys Lys Gly Lys Gly Val Arg Thr Ile Se - #r Ala Pro Thr Asp Arg 
50 - # 55 - # 60 
- - Leu Lys Asp Ile Gln Arg Arg Ile Cys Asp Le - #u Leu Ser Asp Ser Arg 
65 - # 70 - # 75 - # 80 
- - Asp Glu Ile Phe Ala Ile Arg Lys Ile Ser As - #n Asn Tyr Ser Phe Ala 
85 - # 90 - # 95 
- - Phe Glu Arg Gly Lys Ser Ile Ile Leu Asn Al - #a Tyr Lys His Arg Gly 
100 - # 105 - # 110 
- - Lys Gln Ile Ile Leu Asn Ile Asp Leu Lys As - #p Phe Phe Glu Ser Phe 
115 - # 120 - # 125 
- - Asn Phe Gly Arg Val Arg Gly Tyr Phe Leu Se - #r Asn Gln Asp Phe Leu 
130 - # 135 - # 140 
- - Leu Asn Pro Val Val Ala Thr Thr Leu Ala Ly - #s Ala Ala Cys Tyr Asn 
145 1 - #50 1 - #55 1 - 
#60 
- - Gly Thr Leu Pro Gln Gly Ser Pro Cys Ser Pr - #o Ile Ile Ser Asn 
Leu 
165 - # 170 - # 175 
- - Ile Cys Asn Ile Met Asp Met Arg Leu Ala Ly - #s Leu Ala Lys Lys Tyr 
180 - # 185 - # 190 
- - Gly Cys Thr Tyr Ser Arg Tyr Ala Asp Asp Il - #e Thr Ile Ser Thr Asn 
195 - # 200 - # 205 
- - Lys Asn Thr Phe Pro Leu Glu Met Ala Thr Va - #l Gln Pro Glu Gly Val 
210 - # 215 - # 220 
- - Val Leu Gly Lys Val Leu Val Lys Glu Ile Gl - #u Asn Ser Gly Phe Glu 
225 2 - #30 2 - #35 2 - 
#40 
- - Ile Asn Asp Ser Lys Thr Arg Leu Thr Tyr Ly - #s Thr Ser Arg Gln 
Glu 
245 - # 250 - # 255 
- - Val Thr Gly Leu Thr Val Asn Arg Ile Val As - #n Ile Asp Arg Cys Tyr 
260 - # 265 - # 270 
- - Tyr Lys Lys Thr Arg Ala Leu Ala His Ala Le - #u Tyr Arg Thr Gly Glu 
275 - # 280 - # 285 
- - Tyr Lys Val Pro Asp Glu Asn Gly Val Leu Va - #l Ser Gly Gly Leu Asp 
290 - # 295 - # 300 
- - Lys Leu Glu Gly Met Phe Gly Phe Ile Asp Gl - #n Val Asp Lys Phe Asn 
305 3 - #10 3 - #15 3 - 
#20 
- - Asn Ile Lys Lys Lys Leu Asn Lys Gln Pro As - #p Arg Tyr Leu Leu 
Thr 
325 - # 330 - # 335 
- - Asn Ala Thr Leu His Gly Phe Lys Leu Lys Le - #u Asn Ala Arg Glu Lys 
340 - # 345 - # 350 
- - Ala Tyr Thr Lys Phe Ile Tyr Tyr Lys Phe Ph - #e His Gly Asn Thr Cys 
355 - # 360 - # 365 
- - Pro Thr Ile Ile Thr Glu Gly Lys Thr Asp Ar - #g Ile Tyr Leu Lys Ala 
370 - # 375 - # 380 
- - Ala Leu His Ser Leu Glu Thr Ser Tyr Pro Gl - #u Leu Phe Arg Glu Lys 
385 3 - #90 3 - #95 4 - 
#00 
- - Thr Asp Ser Lys Lys Lys Glu Ile Asn Leu As - #n Ile Phe Lys Ser 
Asn 
405 - # 410 - # 415 
- - Glu Lys Thr Lys Tyr Phe Leu Asp Leu Ser Gl - #y Gly Thr Ala Asp Leu 
420 - # 425 - # 430 
- - Lys Lys Phe Leu Glu Arg Tyr Lys Asn Asn Ty - #r Ala Ser Tyr Tyr Ala 
435 - # 440 - # 445 
- - Ser Val Pro Lys His Pro Val Ile Met Leu Le - #u Asp Asn Asp Thr Ala 
450 - # 455 - # 460 
- - Pro Ser Asp Leu Leu Asn Phe Leu Arg Asn Ly - #s Val Lys Ser Cys Pro 
465 4 - #70 4 - #75 4 - 
#80 
- - Asp Asp Val Thr Glu Met Arg Lys Met Lys Ty - #r Ile His Leu Phe 
Tyr 
485 - # 490 - # 495 
- - Asn Leu Tyr Ile Leu Leu Thr Pro Leu Ser Pr - #o Ser Gly Glu Gln Thr 
500 - # 505 - # 510 
- - Ser Met Glu Asp Leu Phe Pro Lys Asp Ile Le - #u Asp Ile Lys Ile Asp 
515 - # 520 - # 525 
- - Gly Lys Lys Phe Asn Lys Asn Asn Asp Gly As - #p Ser Lys Thr Glu Tyr 
530 - # 535 - # 540 
- - Gly Lys His Ile Phe Ser Met Arg Val Val Ar - #g Asp Lys Lys Arg Lys 
545 5 - #50 5 - #55 5 - 
#60 
- - Ile Asp Phe Lys Ala Phe Cys Cys Ile Phe As - #p Ala Ile Lys Asp 
Ile 
565 - # 570 - # 575 
- - Lys Glu His Tyr Lys Leu Met Leu Asn Ser 
580 - # 585 
__________________________________________________________________________