Nucleic acid sequences controlling lung cell-specific gene expression

An oligonucleotide which includes at least one nucleic acid sequence which binds to at least one nuclear protein found in lung cells, such as TTF-1 protein. The oligonucleotide may be contained in a vector. The at least one nuclear protein provides for lung cell-specific expression of the vector upon binding of the at least one nucleic acid sequence to the at least one nuclear protein. Such vector may also include genes encoding therapeutic agents, and may be employed for delivering genes encoding therapeutic agents to lung cells.

This invention relates to nucleic acid sequences which bind to nuclear 
proteins found in lung cells. More particularly, this invention relates to 
nucleic acid sequence(s) which bind to nuclear protein(s) found in lung 
cells, such as TTF-1 protein, and vectors containing said nucleic acid 
sequence(s), whereby lung-specific expression of the vector is effected 
upon binding of said nucleic acid sequence(s) to said nuclear protein(s). 
BACKGROUND OF THE INVENTION 
Lung-specific gene products include the lung surfactant proteins SP-A, 
SP-B, SP-C, SP-D, and Clara cell secretory protein (CCSP). The recent 
cloning of these gene products, the determination of their expression 
patterns in vivo (Weaver, et al., Biochem. J., Vol. 273, page 249-264 
(1991); Wert, et al., Dev. Biol., Vol. 156, pgs. 426-443 (1993); Stripp, 
et al., Genomics, Vol. 20, pgs. 27-35 (1994)); and the characterization of 
cell lines that support their expression (O'Reilly, et al., Biochem. 
Biophys. Acta, Vol. 970, pgs. 194-204 (1988); Gazdar, et al., Cancer Res., 
Vol. 50, pgs. 5481-5487 (1990); Wikenheiser, et al., Proc. Nat. Acad. Sci. 
USA, Vol. 90, pgs. 11029-11033 (1993)) provide a model system to 
investigate the mechanisms involved in lung-specific gene expression. 
The control of tissue-specific gene expression is thought to occur largely 
at the level of transcription initiation. Consistent with this observation 
is that appropriate cis-active sequences from tissue-specific genes often 
are sufficient to target expression of a reporter gene to the tissue of 
origin in vivo. (Jaenisch, Science, Vol. 240, pgs. 1468-1474 (1988).) 
Studies have shown that DNA-binding proteins interact specifically with 
these sequences to stimulate gene transcription (Maniatis, et al., 
Science, Vol. 236, pgs. 1237-1244 (1987): Mitchell, et al., Science, Vol. 
245, pgs. 371-378 (1989); Johnson, et al., Ann. Rev. Biochem., Vol. 58, 
pgs. 799-839 (1989).) Liver-specific cis-active elements have been studied 
extensively, and several transcription factors including HNF-1, HNF-3, 
HNF-4, C/EBP, and DBP (Simmons, et al., Genes & Dev., Vol. 4, pgs. 695-711 
(1990)) bind these regions and appear to act together to regulate 
transcription of liver-specific genes (Costa, et al., Mol. Cell. Biol., 
Vol. 9, pgs. 1415-1425 (1991)). None of these proteins appears to be 
restricted to liver cells. (Xanthopoulus, et al., Proc. Nat. Acad. Sci. 
USA, Vol. 88, pgs. 3807-3811 (1991)). This suggests that mechanisms other 
than the restricted expression of a transcription factor to a single cell 
type are responsible for the tissue-specific activity of these genetic 
elements. This could involve interaction between DNA bound factors at a 
unique cis-active environment (Milos, et al, Genes and Dev., Vol. 6, pgs. 
991-1004 (1992); Nerlov, et al., Genes and Dev., Vol. 8, pgs. 350-362 
(1994)) or between a DNA bound factor and a non-DNA bound cofactor 
(Mendel, et al., J. Biol. Chem., Vol. 266, pgs. 677-680 (1991)). 
Recently, it has appeared that the mechanisms of transcriptional control of 
tissue-specific genes in the liver and lung may be related. This is 
suggested by the expression of HNF-3 and CCAAT enhancer binding 
protein-.alpha. (C/EBP) family members in the lung, (Lai, et al., Genes 
and Dev., Vol. 5, pgs. 416-427 (1991); Cao, et al., Genes & Dev., Vol. 5, 
pgs. 1538-1552 (1991); Xanthopoulus, et al., 1991), and by the finding 
that HNF-3 proteins bind to a region of the CCSP gene promoter in vitro 
(Sawaya, et al., Mol. Cell. Biol., Vol. 13, pgs. 3860-3871 (1993); Bingle, 
et al; Biochem J., Vol. 295, pgs. 227-232 (1993)). 
Despite the work accomplished in the above studies, a need still exists to 
isolate and obtain genetic elements which will direct lung cell specific 
expression of genes of interest. 
DETAILED DESCRIPTION OF THE INVENTION 
In accordance with an aspect of the present invention, there is provided an 
oligonucleotide or polynucleotide including at least one nucleic acid 
sequence which binds to at least one nuclear protein found in lung cells. 
The term "nucleic acid sequence" as used herein, means a DNA or RNA 
molecule, and more particularly a linear series of deoxyribonucleotides or 
ribonucleotides connected one to the other by phosphodiester bonds between 
the 3' and 5' carbons of the adjacent pentoses. Depending upon the use 
herein, such term includes complete and partial gene sequences, and 
includes polynucleotides as well. 
In a preferred embodiment, the at least one nucleic acid sequence which 
binds to a nuclear protein found in lung cells is contained in the 
proximal promoter region of the human surfactant protein B (or SP-B) gene. 
Such proximal promoter region is found from base-218 to base +41 of the 
human surfactant protein B gene. In one embodiment, the at least one 
nucleic acid sequence which binds to a nuclear protein found in lung cells 
is contained in a portion of the proximal promoter region of the human 
surfactant protein B gene, as defined by the region from base -118 to base 
-64 of the human surfactant protein B gene. 
In another embodiment, the at least one nucleic acid sequence which binds 
to a nuclear protein found in lung cells is contained in a portion of the 
proximal promoter region of the human surfactant protein B gene, as 
defined by the region from base -111 to base -73 of the human surfactant 
protein B gene. 
In another embodiment, the at least one nucleic acid sequence which binds 
to a nuclear protein found in lung cells is contained in the distal 
promoter region of the human surfactant protein B (or SP-B) gene. Such 
distal promoter region is found from base -439 to base -331 of the human 
surfactant protein B gene. In one embodiment, the at least one nucleic 
acid sequence which binds to a nuclear protein found in lung cells is 
contained in one or more portions of the distal promoter region of the 
human surfactant protein B gene, as defined by the regions from (i) base 
-439 to base -410; or (ii) base -417 to base -390; or (iii) base -396 to 
base -367 of the human surfactant protein B gene. 
Applicants have found that such proximal promoter region and distal 
promoter region of the human SP-B gene contain enhancer-like elements. 
Such enhancer-like elements may bind to nuclear proteins found 
specifically in lung cells, or to ubiquitous nuclear proteins (i.e., 
nuclear proteins found in lung cells as well as other cell types). The 
binding of such enhancer-like elements to nuclear proteins in lung cells 
enables one to express genes specifically in lung cells transduced with 
vectors including such enhancer-like elements. 
In another embodiment, the at least one nucleic acid sequence which binds 
to a nuclear protein found in lung cells is contained in the promoter 
region of the CCSP protein gene. In yet another embodiment, the at least 
one nucleic acid sequence which binds to a nuclear protein found in lung 
cells is contained in the promoter region of the mouse surfactant protein 
C (SP-C) gene. 
In yet another embodiment, the at least one nucleic acid sequence which 
binds to a nuclear protein found in lung cells is contained in a portion 
of the promoter region of the mouse surfactant protein C (SP-C) gene, as 
defined by the region from base -180 to base -160 of the mouse surfactant 
protein C gene. 
In another embodiment, the at least one nucleic acid sequence which binds 
to a nuclear protein found in lung cells is contained in the promoter 
region of the human surfactant protein C (or SP-C) gene. In one 
embodiment, the at least one nucleic acid sequence which binds to a 
nuclear protein found in lung cells is contained in a portion of the 
promoter region of the human SP-C gene as defined by the region from base 
-180 to base -160 of the human SP-C gene. 
In another embodiment, the at least one nucleic acid sequence which binds 
to a nuclear protein found in lung cells is contained in a portion of the 
promoter region of the mouse surfactant protein A (SP-A) gene as defined 
by the region from base -255 to base -57 of the mouse SP-A gene. 
In yet another embodiment, the at least one nucleic acid sequence which 
binds to a nuclear protein found in lung cells is contained in a portion 
of the promoter region of the mouse surfactant protein A (SP-A) gene as 
defined by the region from base -231 to base -168 of the mouse SP-A gene. 
In a further embodiment, the at least one nucleic acid sequence which binds 
to a nuclear protein found in lung cells is contained in the distal 
promoter region of the mouse surfactant protein B (SP-B) gene. In one 
embodiment, the at least one nucleic acid sequence which binds to a 
nuclear protein found in lung cells is contained in one or more portions 
of the distal promoter region of the mouse surfactant protein B gene, as 
defined by the regions from (i) base -345 to base -331; or (ii) base -370 
to base -356; or (iii) base -332 to base -318; or (iv) base -296 to base 
-282 of the mouse surfactant protein B gene. 
In another embodiment, the at least one nucleic acid sequence which binds 
to a nuclear protein found in lung cells is contained in the proximal 
promoter region of the mouse surfactant protein B gene. In one embodiment, 
the at least one nucleic acid sequence which binds to a nuclear protein 
found in lung cells is contained in a portion of the proximal promoter 
region of the mouse surfactant protein B gene as defined by the region 
from base -18 to base -5 of the mouse surfactant protein B gene. 
In another embodiment, the oligonucleotide includes at least one nucleic 
acid sequence which binds to thyroid transcription factor-1, or TTF-1 
protein. TTF-1 protein is described further in Francis-Lang, et al., Mol. 
Cell. Biol., Vol. 12, No. 2, pgs. 576-588 (February 1992). The DNA 
sequence encoding human TTF-1 protein is described in Ikeda, et al., J. 
Biol. Chem., Vol. 270, No. 14, pgs. 8108-8114 (Apr. 7, 1995). 
In a preferred embodiment, the at least one nucleic acid sequence which 
binds to TTF-1 protein includes a nucleic acid sequence, also known as a 
"core" nucleic acid sequence, which binds to TTF-1 protein, and which has 
the following structure: 
WXNNYZ. 
W is cytosine, guanine, or thymine. X is cytosine, thymine, or adenine. N 
is adenine, cytosine, guanine, or thymine. Y is adenine, thymine, or 
guanine. Z is guanine, adenine, or cytosine. 
In one embodiment, W is cytosine. In another embodiment, X is thymine. In 
yet another embodiment, X is cytosine. 
In yet another embodiment, Y is adenine, and in a further embodiment, Z is 
guanine. In another embodiment, Z is cytosine. 
In a most preferred embodiment, the nucleic acid sequence has the following 
structure: 
CTNNAG. 
In another embodiment, the nucleic acid sequence which binds to TTF-1 
protein may be one of the following: 
CTGGAG (SEQ. ID NO.: 1); 
CTTCAG (SEQ. ID NO.: 2); 
CTCATA (SEQ. ID NO.: 3); 
GCCAAG (SEQ. ID NO.: 4); 
CTCAAG (SEQ. ID NO.: 5); 
CTCCAG (SEQ. ID NO.: 6); 
GTCAAG (SEQ. ID NO.: 7); 
TCTAAG (SEQ. ID NO.: 8); 
GTTAAG (SEQ. ID NO.: 9); 
CTGAAG (SEQ. ID NO.: 10); 
TCCAGG (SEQ. ID NO.: 11); 
CCGAAC (SEQ. ID NO.: 12); 
CCCAAG (SEQ. ID NO.: 13); 
CATAAG; (SEQ. ID NO.: 14) or 
TAGAGA (SEQ. ID NO.: 15). 
Such "core" nucleic acid sequences, in general, are contained within larger 
nucleic acid sequences or oligonucleotides. Representative examples of 
nucleic acid sequences or oligonucleotides which include the above "core" 
sequences include the following: 
(a) TCAAGCACCTGGAGGGCTCT (SEQ. ID NO.: 16); 
(b) GGAGGGCTCTTCAGAGCAAA (SEQ. ID NO.: 17); 
(c) AGGTGCCACTCATAGAAAGC (SEQ. ID NO.: 18); 
(d) TTGTTTCTGCCAAGTGCTGG (SEQ. ID NO.: 19); 
(e) GATGCCCACTCAAGCTTAGA (SEQ. ID NO.: 20); 
(f) GGTGACCACTCCAGGACATG (SEQ. ID NO.: 21); 
(g) ACTGATTACTCAAGTATTCT (SEQ. ID NO.: 22); 
(h) GGAGCAGACTCAAGTAGAGG (SEQ. ID NO.: 23); 
(i) ACTGCCCAGTCAAGTGTTCT; (SEQ. ID NO.: 24) and 
(j) AGCACCTGGAGGGCTCTTCAGAGC (SEQ. ID NO.: 25). 
Sequence (j), which the Applicants refer to as the SPB-f1 site, is 
contained in the proximal promoter region of the human lung surfactant 
protein B gene, and will be described further hereinbelow. 
In yet another preferred embodiment, the at least one nucleic acid sequence 
which binds to TTF-1 protein includes the "core" nucleic acid sequence: 
CAAG. 
Representative examples of such nucleic acid sequences include, but are not 
limited to, those hereinabove described. 
Although the scope of the present invention is not to be limited to any 
theoretical reasoning, Applicants have found that the above nucleic acid 
sequences, which may be found in the promoter region of the lung 
surfactant protein B gene, and include a "core" nucleic acid sequence 
which binds to TTF-1 protein (thyroid transcription factor 1 protein), 
activates expression of the lung surfactant protein gene by virtue of the 
binding of the "core", nucleic acid sequence to TTF-1 protein. Applicants 
also have discovered that such nucleic acid sequences also may be employed 
in order to direct expression of genes encoding proteins other than lung 
surfactant proteins in lung cells. 
In another embodiment, the oligonucleotide further includes a sequence 
which binds to HNF-3 protein. Although HNF-3 protein is not found 
exclusively in lung tissue, Applicants have found that when a nucleic acid 
sequence which binds to HNF-3 protein is located in proximity to the 
nucleic acid sequence(s) which bind to TTF-1 protein, one obtains improved 
lung-specific expression of any nucleic acid sequences contained in 
vectors including the nucleic acid sequences which bind to TTF-1 protein 
and which bind to HNF-3 protein. HNF-3 protein is described further in 
Overdier, et al., Mol. Cell. Biol., Vol. 14, No. 4 (April 1994). 
In one embodiment, the nucleic acid sequence which binds to HNF-3 protein 
includes a nucleic acid sequence having the following structure: 
BADTETTFEDTD (SEQ. ID NO.: 26), 
wherein B is adenine, cytosine, or guanine; D is adenine, thymine, or 
uracil; E is adenine or guanine; and F is guanine, thymine, or uracil. 
Preferably, the nucleic acid sequence which binds to HNF-3 protein 
includes a nucleic acid sequence having one of the following structures: 
(a) CAGTGTTTGCCT; (SEQ. ID NO.: 27) or 
(b) GCAAAGACAAACACTGAGG (SEQ. ID NO.: 28). 
Sequence (b), which the Applicants refer to as the SPB-f2 site, is found in 
the proximal promoter region of the human lung surfactant protein B gene, 
and will be described further hereinbelow. 
In another embodiment, the oligonucleotide further includes a sequence 
which binds to HNF-5 protein. 
As stated hereinabove, the oligonucleotides of the present invention, which 
contain the nucleic acid sequences(s) which bind(s) to nuclear proteins 
found in lung cells, may be employed in order to direct expression of 
genes encoding lung surfactant proteins, as well as other proteins, in 
lung cells. Thus, such oligonucleotides may be contained in an appropriate 
vector. Upon binding of the at least one nucleic acid sequence to the at 
least one nuclear protein found in lung cells, lung-specific expression of 
the vector is provided. 
The term "vector" as used herein, means an agent containing or consisting 
of a DNA or RNA capable of introducing a nucleic acid sequence(s) into a 
cell, resulting in the expression of the nucleic acid sequence(s) in the 
cell. 
Such vectors include, but are not limited to, eukaryotic or prokaryotic 
plasmids (such as, for example, bacterial plasmids), and viral vectors. 
The vector also may be contained within a liposome. 
Such vectors, which include a nucleic acid sequence(s) which binds to TTF-1 
protein, and which also may include a nucleic acid sequence which binds to 
HNF-3 protein, may also include at least one nucleic acid sequence 
encoding a therapeutic agent, whereby such vectors enable the expression 
of therapeutic agents in lung cells. 
The term "therapeutic" is used in a generic sense and includes treating 
agents, prophylactic agents, and replacement agents. 
In one embodiment, the vector is a viral vector. Viral vectors which may be 
employed include, but are not limited to, retroviral vectors, adenovirus 
vectors, adeno-associated virus vectors, and Herpes Virus vectors. 
The adenoviral vector which is employed may, in one embodiment, be an 
adenoviral vector which includes essentially the complete adenoviral 
genome (Shenk, et al., Curr. Top. Microbiol. Immunol., 111(3): 1-39 
(1984)). Alternatively, the adenoviral vector may be a modified adenoviral 
vector in which at least a portion of the adenoviral genome has been 
deleted. 
In one embodiment, the adenoviral vector comprises an adenoviral 5' ITR; an 
adenoviral 3' ITR; an adenoviral encapsidation signal; a DNA sequence 
which binds to TTF-1 protein, a DNA sequence which binds to HNF-3 protein, 
and at least one DNA sequence encoding a therapeutic agent. The vector is 
free of at least the majority of adenoviral E1 and E3 DNA sequences, but 
is not free of all of the E2 and E4 DNA sequences, and DNA sequences 
encoding adenoviral proteins promoted by the adenoviral major late 
promoter. 
In still another embodiment, the gene in the E2a region that encodes the 72 
kilodalton binding protein is mutated to produce a temperature sensitive 
protein that is active at 32.degree. C., the temperature at which the 
viral particles are produced. This temperature sensitive mutant is 
described in Ensinger, et al., J. Virology, 10:328-339 (1972), Van der 
Vliet, et al., J. Virology, 15:348-354 (1975), and Friefeld, et al., 
Virology, 124:380-389 (1983). 
In yet another embodiment, the vector is free of at least the majority of 
the E1 and E3 DNA sequences, is free of at least a portion of at least one 
DNA sequence selected from the group consisting of the E2 and E4 DNA 
sequences, and is free of DNA sequences encoding adenoviral proteins 
promoted by the adenoviral major late promoter. 
Such a vector, in a preferred embodiment, is constructed first by 
constructing, according to standard techniques, a shuttle plasmid which 
contains, beginning at the 5' end, the "critical left end elements," which 
include an adenoviral 5' ITR, an adenoviral encapsidation signal, and an 
E1a enhancer sequence; a promoter (which may be an adenoviral promoter or 
a foreign promoter); a multiple cloning site (which may be as hereinabove 
described); a poly A signal; and a DNA segment which corresponds to a 
segment of the adenoviral genome. The vector also may contain a tripartite 
leader sequence. The DNA segment corresponding to the adenoviral genome 
serves as a substrate for homologous recombination with a modified or 
mutated adenovirus, and such sequence may encompass, for example, a 
segment of the adenovirus 5 genome no longer than from base 3329 to base 
6246 of the genome. The plasmid may also include a selectable marker and 
an origin of replication. The origin of replication may be a bacterial 
origin of replication. Representative examples of such shuttle plasmids 
include pAVS6, shown in FIG. 19. The DNA including the DNA sequence which 
binds to the nuclear protein found in lung cells, such as TTF-1 protein, 
and may also include a DNA sequence which binds to HNF-3 protein or which 
binds to HNF-5 protein, and the DNA encoding therapeutic agent may be 
inserted into the multiple cloning site as a "cassette," or such elements 
may be inserted in separate cloning steps. One may amplify the expression 
of the DNA encoding the therapeutic agent by adding to the plasmid 
increased numbers of cassettes or of the DNA sequence which binds to the 
nuclear protein found in lung cells, such as TTF-1 protein. 
This construct is then used to produce an adenoviral vector. Homologous 
recombination is effected with a modified or mutated adenovirus in which 
at least the majority of the E1 and E3 adenoviral DNA sequences have been 
deleted. Such homologous recombination may be effected through 
co-transfection of the plasmid vector and the modified adenovirus into a 
helper cell line, such as 293 cells, by CaPO.sub.4 precipitation. Upon 
such homologous recombination, a recombinant adenoviral vector is formed 
that includes DNA sequences derived from the shuttle plasmid between the 
NotI site and the homologous recombination fragment, and DNA derived from 
the E1 and E3 deleted adenovirus between the homologous recombination 
fragment and the 3' ITR. 
In one embodiment, the homologous recombination fragment overlaps with 
nucleotides 3329 to 6246 of the adenovirus 5 (ATCC VR-5) genome. 
Through such homologous recombination, a vector is formed which includes an 
adenoviral 5' ITR, an adenoviral encapsidation signal; an E1a enhancer 
sequence; a promoter; at least one DNA sequence which binds to a nuclear 
protein found in lung cells, such as TTF-1 protein; and may also include 
at least one DNA sequence which binds HNF-3 protein or HNF-5 protein; at 
least the DNA sequence which encodes a therapeutic agent; a poly A signal; 
adenoviral DNA free of at least the majority of the E1 and E3 adenoviral 
DNA sequences; and an adenoviral 3' ITR. The vector also may include a 
tripartite leader sequence. This vector may then be transfected into a 
helper cell line, such as HeLa cells, or the 293 helper cell line (ATCC 
No. CRL1573), which will include the E1a and E1b DNA sequences, which are 
necessary for viral replication, and to generate infectious adenoviral 
particles. Transfection may take place by electroporation, calcium 
phosphate precipitation, microinjection, or through proteoliposomes, H441 
cells (ATCC catalog no. HTB-174) may be employed to test for cell 
specificity. 
The vector hereinabove described may include a multiple cloning site to 
facilitate the insertion of DNA sequence(s) into the cloning vector. 
In general, the multiple cloning site includes "rare" restriction enzyme 
sites; i.e., sites which are found in eukaryotic genes at a frequency of 
from about one in every 10,000 to about one in every 100,000 base pairs. 
An appropriate vector in accordance with the present invention is thus 
formed by cutting the cloning vector by standard techniques at appropriate 
restriction sites in the multiple cloning site, and then ligating the DNA 
sequence encoding a therapeutic agent into the cloning vector. 
The infectious viral particles then may be administered to a host, whereby 
the infectious viral particles will infect lung cells. The viral particles 
are administered in an amount effective to produce a therapeutic effect in 
a host. In one embodiment, the viral particles may be administered in an 
amount of from about 10.sup.6 to about 10.sup.12 plaque forming units 
(pfu), preferably from about 10.sup.9 to about 10.sup.11 pfu. The host may 
be a human or non-human animal host. 
Preferably, the infectious viral vector particles are administered 
systemically, such as, for example, by intranasal or intratracheal 
administration. The viral vector particles also may be administered 
intravenously, intraperitoneally, or endotracheally, suspended in normal 
saline or phosphate buffered saline (pH 7.0). 
The vector particles may be administered in combination with a 
pharmaceutically acceptable carrier suitable for administration to a 
patient. The carrier may be a liquid carrier (for example, a saline 
solution), or a solid carrier, such as, for example, microcarrier beads. 
As an alternative to constructing an adenoviral vector particle, an 
adenoviral vector may be constructed as hereinabove described, and then 
encapsulated into liposomes, or complexed with lipids such as lipofectins 
or cytofectins. The adenoviral vector which is contained within a liposome 
or coupled to a lipid may be administered to a host as hereinabove 
described. The preparation of liposomes which contain the adenoviral 
vector, and the coupling of the adenoviral vector to a lipid are known to 
those skilled in the art. Examples of liposomes which may be employed 
include but are not limited to, those disclosed in U.S. Pat. No. 
4,394,448, Nicolau, et al. Proc. Nat. Acad. Sci., Vol. 80, pg. 1068 
(1983), and Nabel, et al., Proc. Nat. Acad. Sci., Vol. 90, pgs. 
11307-11311 (December 1993). Examples of lipofectins which may be employed 
include any protein or polypeptide having a therapeutic effect. Such 
protection or polypeptides include, but are not limited to, those 
disclosed in Felgner, et al., Proc. Nat. Acad. Sci., Vol. 8, pg. 7413 
(1987). Examples of cytofectins which may be employed include, but are not 
limited to, those disclosed in U.S. Pat. No. 5,264,618. 
Therapeutic agents which may be encoded by a DNA or RNA sequence(s) placed 
in the vector include, any protein or polypeptide having a therapeutic 
effect. Such proteins or polypeptides include, but are not limited to, 
those encoded by DNA or RNA sequences encoding lung surfactant proteins, 
such as SP-A, SP-B, SP-C, and SP-D for protection from lung injury; Clara 
Cell Secretory Protein (CCSP); the .alpha.-1-antitrypsin gene for treating 
lung fibrosis, cystic fibrosis, or emphysema; the cystic fibrosis 
transmembrane conductance regulator (CFTR); antioxidants such as, but not 
limited to, manganese superoxide dismutase (Mn-SOD), catalase, 
copper-zinc-superoxide dismutase (CuZn-SOD), extracellular superoxide 
dismutase (EC-SOD), and glutathione reductase, for treatment of acute lung 
injury, oxygen injury, or after chemical exposure to oxidants, infectious 
agents, shock, or for protection of the normal lung during chemotherapy 
for tumors (using bleomycin, adriamycin, or radiation); clotting factors, 
such as Factor VIII and Factor IX; and anti-tumor agents, such as, but not 
limited to, the Herpes Simplex thymidine kinase gene, wherein tumor 
killing is initiated by therapy with gancyclovir or acyclovir; GM-CSF 
(granulocyte-macrophage colony stimulating factor) which also may treat 
alveolar proteinosis, and cytokines such as TNF-.alpha. or Interleukin-1; 
and growth factors such as epidermal growth factor (EGF), and keratinocyte 
growth (KGF), for repair of or protection from injury after infection or 
oxygen therapy, bronchopulmonary dysplasia, or after therapy with lung 
oxidants such as antitumor agents, paraquot toxicity, or after exposure to 
toxins (e.g., alkylating agents, chemical warfare agents) or lung burns. 
In addition, the vector may include antisense DNA or RNA sequences. 
Promoters which may control the genes encoding the therapeutic agents 
include may be promoters which include the nucleic acid sequence(s) which 
bind to the nuclear protein(s) bound in lung cells. Alternatively, the 
promoter may be a homologous or heterologous promoter. Such promoters 
include, but are not limited to, human globin promoters; viral thymidine 
kinase promoters, such as the Herpes Simplex thymidine kinase promoter; 
adenoviral late terminal repeats; retroviral LTRs; surfactant protein A, 
B, or C (SP-A, SP-B, or SP-C) promoters; the Clara Cell secretory protein 
(CCSP) promoter; the .beta.-actin promoter; and human growth hormone 
promoters. The promoter also may be the native promoter which controls the 
gene encoding the therapeutic agent. In general, the promoter will include 
a TATA box, transcription start signal, and a CAAT box or variation 
thereof. 
For example, one may construct a vector in accordance with the present 
invention which includes the CFTR gene. The vector then may be 
administered to the respiratory epithelium in an effective therapeutic 
amount for the correction of the pulmonary deficit in patients with cystic 
fibrosis. In another example, vectors containing functional proteins may 
be delivered to the respiratory epithelium in order to correct 
deficiencies in such proteins. Such functional proteins include 
antioxidants, .alpha.-1-antitrypsin, CFTR, lung surfactant proteins, 
cytokines, and growth factors such as EGF and KGF, and may also include 
adenosine deaminase for treatment of severe combined immune deficiency, 
von Willebrand's factor for treatment of Christmas disease, and 
.beta.-glucuronidase for treatment of Gaucher's disease. Also, vectors 
including genes encoding anti-cancer agents or anti-inflammatory agents 
may be administered to lung cells of a patient for the treatment of lung 
cancer or inflammatory lung disease. 
In another embodiment, the viral vector is a retroviral vector. 
Examples of retroviral vectors which may be employed include, but are not 
limited to, Moloney Murine Leukemia Virus, spleen necrosis virus, and 
vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey 
Sarcoma Virus, avian leukosis virus, gibbon ape leukemia virus, human 
immunodeficiency virus, myeloproliferative sarcoma virus, and mammary 
tumor virus. Preferably, the retroviral vector is an infectious but 
non-replication competent retrovirus; however, replication competent 
retroviruses may also be used. 
Retroviral vectors are useful as agents to mediate retroviral-mediated gene 
transfer into eukaryotic cells. Retroviral vectors generally are 
constructed such that the majority of sequences coding for the structural 
genes of the virus are deleted and replaced by the gene(s) of interest. 
Most often, the structural genes (i.e., gag, pol, and env), are removed 
from the retroviral backbone using genetic engineering techniques known in 
the art. This may include digestion with the appropriate restriction 
endonuclease or, in some instances, with Bal 31 exonuclease to generate 
fragments containing appropriate portions of the packaging signal. 
These new genes have been incorporated into the proviral backbone in 
several general ways. The most straightforward constructions are ones in 
which the structural genes of the retrovirus are replaced by a single gene 
which then is transcribed under the control of the viral regulatory 
sequences within the long terminal repeat (LTR). Retroviral vectors have 
also been constructed which can introduce more than one gene into target 
cells. Usually, in such vectors one gene is under the regulatory control 
of the viral LTR, while the second gene is expressed either off a spliced 
message or is under the regulation of its own, internal promoter. 
Efforts have been directed at minimizing the viral component of the viral 
backbone, largely in an effort to reduce the chance for recombination 
between the vector and the packaging-defective helper virus within 
packaging cells. A packaging-defective helper virus is necessary to 
provide the structural genes of a retrovirus, which have been deleted from 
the vector itself. 
In one embodiment, the retroviral vector may be one of a series of vectors 
described in Bender, et al., J. Virol. 61:1639-1649 (1987), based on the 
N2 vector (Armentano, et al., J. Virol., 61:1647-1650) containing a series 
of deletions and substitutions to reduce to an absolute minimum the 
homology between the vector and packaging systems. These changes have also 
reduced the likelihood that viral proteins would be expressed. In the 
first of these vectors, LNL-XHC, there was altered, by site-directed 
mutagenesis, the natural ATG start codon of gag to TAG, thereby 
eliminating unintended protein synthesis from that point. In Moloney 
murine leukemia virus (MoMuLV), 5' to the authentic gag start, an open 
reading frame exists which permits expression of another glycosylated 
protein (pPr80.sup.gag). Moloney murine sarcoma virus (MoMuSV) has 
alterations in this 5' region, including a frameshift and loss of 
glycosylation sites, which obviate potential expression of the amino 
terminus of pPr80.sup.gag. Therefore, the vector LNL6 was made, which 
incorporated both the altered ATG of LNL-XHC and the 5' portion of MoMuSV. 
The 5' structure of the LN vector series thus eliminates the possibility 
of expression of retroviral reading frames, with the subsequent production 
of viral antigens in genetically transduced target cells. In a final 
alteration to reduce overlap with packaging-defective helper virus, Miller 
has eliminated extra env sequences immediately preceding the 3' LTR in the 
LN vector (Miller, et al., Biotechniques, 7:980-990, 1989). 
The paramount need that must be satisfied by any gene transfer system for 
its application to gene therapy is safety. Safety is derived from the 
combination of vector genome structure together with the packaging system 
that is utilized for production of the infectious vector. Miller, et al. 
have developed the combination of the pPAM3 plasmid (the 
packaging-defective helper genome) for expression of retroviral structural 
proteins together with the LN vector series to make a vector packaging 
system where the generation of recombinant wild-type retrovirus is reduced 
to a minimum through the elimination of nearly all sites of recombination 
between the vector genome and the packaging-defective helper genome (i.e. 
LN with pPAM3). 
In one embodiment, the retroviral vector may be a Moloney Murine Leukemia 
Virus of the LN series of vectors, such as those hereinabove mentioned, 
and described further in Bender, et al. (1987) and Miller, et al. (1989). 
Such vectors have a portion of the packaging signal derived from a mouse 
sarcoma virus, and a mutated gag initiation codon. The term "mutated" as 
used herein means that the gag initiation codon has been deleted or 
altered such that the gag protein or fragments or truncations thereof, are 
not expressed. 
In another embodiment, the retroviral vector may include at least four 
cloning, or restriction enzyme recognition sites, wherein at least two of 
the sites have an average frequency of appearance in eukaryotic genes of 
less than once in 10,000 base pairs; i.e., the restriction product has an 
average DNA size of at least 10,000 base pairs. Preferred cloning sites 
are selected from the group consisting of NotI, SnaBI, SalI, and XhoI. In 
a preferred embodiment, the retroviral vector includes each of these 
cloning sites. Such vectors are further described in U.S. Pat. No. 
5,672,510 and incorporated herein by reference in its entirety. 
When a retroviral vector including such cloning sites is employed, there 
may also be provided a shuttle cloning vector which includes at least two 
cloning sites which are compatible with at least two cloning sites 
selected from the group consisting of NotI, SnaBI, SalI, and XhoI located 
on the retroviral vector. The shuttle cloning vector also includes at 
least one desired gene which is capable of being transferred from the 
shuttle cloning vector to the retroviral vector. 
The shuttle cloning vector may be constructed from a basic "backbone" 
vector or fragment to which are ligated one or more linkers which include 
cloning or restriction enzyme recognition sites. Included in the cloning 
sites are the compatible, or complementary cloning sites hereinabove 
described. Genes and/or promoters having ends corresponding to the 
restriction sites of the shuttle vector may be ligated into the shuttle 
vector through techniques known in the art. 
The shuttle cloning vector can be employed to amplify DNA sequences in 
prokaryotic systems. The shuttle cloning vector may be prepared from 
plasmids generally used in prokaryotic systems and in particular in 
bacteria. Thus, for example, the shuttle cloning vector may be derived 
from plasmids such as pBR322; pUC 18; etc. 
The vector includes one or more promoters. Suitable promoters which may be 
employed include, but are not limited to, the retroviral LTR; the SV40 
promoter; and the human cytomegalovirus (CMV) promoter described in 
Miller, et al., Biotechniques, Vol. 7, No. 9, 980-990 (1989), or any other 
promoter (e.g., cellular promoters such as eukaryotic cellular promoters 
including, but not limited to, the histone, pol III, and .beta.-actin 
promoters). Other viral promoters which may be employed include, but are 
not limited to, adenovirus promoters, TK promoters, and B19 parvovirus 
promoters. The selection of a suitable promoter will be apparent to those 
skilled in the art from the teachings contained herein. These promoters 
may be altered, by deletion mutation(s), to provide a basic transcription 
unit that can be modified by the addition of the TTF-1 binding cis-acting 
sequence. 
The vector then is employed to transduce packaging cell lines to form 
producer cell lines. Examples of packaging cells which may be transfected 
include, but are not limited to, the PE501, 17, .psi.-2, .psi.-AM, 
2, T19-14X, VT-19-17-H2, .psi.CRE, .psi.CRIP, GP+E-86, GP+envAm12, and 
DAN cell lines as described in Miller, Human Gene Therapy, Vol. 1, pgs. 
5-14 (1990), which is incorporated herein by reference in its entirety. 
The vector may transduce the packaging cells through any means known in 
the art. Such means include, but are not limited to, electroporation, the 
use of liposomes, such as hereinabove described, and CaPO.sub.4 
precipitation. In one alternative, the retroviral plasmid vector may be 
encapsulated into a liposome, or coupled to a lipid, as hereinabove 
described, and then administered to a host, also as hereinabove described. 
The producer cell line generates infectious but non-replicating viral 
vector particles which include the nucleic acid sequence(s) which bind(s) 
to a nuclear protein found in lung cells, such as to TTF-1 protein, and 
may also include nucleic acid sequence(s) which bind(s) to HNF-3 protein 
or HNF-5 protein, and the nucleic acid sequence(s) encoding a therapeutic 
agent. Such vector particles then may be employed to transduce lung cells, 
which will express the nucleic acid sequence(s) encoding the therapeutic 
agent(s). The vector particles may transduce the lung cells at a 
multiplicity of infection of from 0.1 to 100 vectors per cell, preferably 
from 1 to 10 vectors per cell, and more preferably at about 10 vectors per 
cell. 
Therapeutic agents which may be encoded by at least one nucleic acid 
sequence contained in the viral vector particles may be those as 
hereinabove described. The vector also may include an antisense DNA or RNA 
sequence. Promoters controlling such nucleic acid sequences also may be 
those hereinabove described. 
In a preferred embodiment, DNA binding sites for thyroid transcription 
factor 1 (TTF-1) alone or in combination with hepatocyte nuclear factor 3 
(HNF-3) are used to direct lung specific transcription of a therapeutic 
gene or cDNA. This may be accomplished by using TTF-1 and HNF-3 DNA 
binding sites in some combination with a minimal homologous or 
heterologous promoter. This transcription unit could be linked to a 
therapeutic cDNA or gene, introduced into a plasmid or viral DNA 
(adenoviral, retroviral, adeno-associated or other viral vector) vector, 
and delivered systemically or locally to achieve lung-specific 
transcription of the linked therapeutic cDNA or gene. The use of TTF-1 and 
HNF-3 binding sites in the transcription unit of DNA-based gene delivery 
vectors allows a specific therapeutic gene product to be expressed only in 
lung epithelial cells that contain TTF-1 and HNF-3 regulatory factors even 
when the vector was delivered systemically, since the TTF-1 component of 
the vector will support gene transcription in a highly lung selective 
manner. This vector could be delivered systemically, or via the trachea, 
without the complication of ectopic expression outside of the lung. In 
addition, more precise regulation of the therapeutic gene could be 
achieved by use of known lung-specific genetic elements such as from the 
SP-B gene. This could involve delivery of the cystic fibrosis 
transmembrane conductance regulator (CFTR) to the respiratory epithelium 
for correction of the pulmonary deficit in patients with cystic fibrosis, 
or replacement of functional proteins in the respiratory epithelium or 
local lung-specific production of a toxic drug for treatment of lung 
cancer or inflammatory lung disease. Protein (gene products) be directed 
for secretion into the airway or the systemic circulation. For example, 
.alpha.-1-antitrypsin cytokines (GM-CSF), intracellular proteins 
(antioxidant genes), CFTR or circulating proteins (clotting factors) could 
be expressed in lung epithelial cells with the lung selective DNA binding 
sites for therapy of common pulmonary and non-pulmonary diseases. 
The nucleic acid sequences of the present invention also may be used as 
probes to detect cancer which has originated in the lung or thyroid. The 
probes are prepared by techniques known to those skilled in the art. 
Because TTF-1 protein and HNF-3 protein are found in cells of cancers 
which originate in the lung or thyroid, one may obtain a sample of cancer 
cells from a patient and contact such cells with a nucleic acid sequence 
which includes at least one nucleic acid sequence which binds to TTF-1 
protein (and preferably also includes at least one nucleic acid sequence 
which binds to HNF-3 protein). Binding of the nucleic acid sequence to the 
cancer cells then is determined by standard techniques. If the nucleic 
acid sequence binds to the cancer cells, then one would know that the 
cancer originated in the lung or thyroid. Once one determines whether the 
cancer originated in the lung or thyroid, an appropriate course of 
treatment of the cancer then may be undertaken. 
In addition, the nucleic acid sequence which binds to TTF-1 protein (and 
preferably also binds to HNF-3 protein) may be placed into a vector which 
also includes a negative selective marker, such as, for example, the 
Herpes Simplex thymidine kinase gene. In one embodiment, the vector is a 
retroviral vector. Such a retroviral vector then may be administered to a 
patient suffering from cancer which has originated in the lung. Upon 
administration of the vector, the vector infects the cancer cells. After 
infection of the cancer cells with the vector, an interaction agent is 
administered to the patient. The interaction agent, such as, for example, 
ganciclovir, interacts with the Herpes Simplex thymidine kinase expressed 
in the cancer cells, whereby such cancer cells are killed. 
In accordance with another aspect of the present invention, there is 
provided a method of detecting cancer which has originated in the lung. 
The method comprises obtaining a sample of cancer cells from a patient, 
and contacting the cancer cells with at least one antibody which 
recognizes an epitope of a protein selected from the group consisting of 
nuclear proteins found in lung cells and lung surfactant proteins. Binding 
of the at least one antibody to the cancer cells then is determined. The 
antibody may be a polyclonal or monoclonal antibody. 
In one embodiment, the at least one antibody recognizes an epitope of a 
nuclear protein found in lung cells. 
Nuclear proteins to which the at least one antibody may bind include, but 
are not limited to, TTF-1 protein. 
In another embodiment, the at least one antibody recognizes an epitope of a 
lung surfactant protein. Lung surfactant proteins to which the at least 
one antibody may bind include, but are not limited to, surfactant protein 
A (SP-A) and surfactant protein B (SP-B). 
Cancers originating in the lung which may be detected include, but are not 
limited to, lung adenocarcinomas, squamous cell lung carcinomas, and small 
cell lung carcinomas. 
In accordance with yet another aspect of the present invention, there is 
provided an isolated polynucleotide comprising a member selected from the 
group consisting of: (a) a polynucleotide encoding human TTF-1 protein; 
(b) a polynucleotide which is substantially homologous to the 
polynucleotide of (a); (c) a polynucleotide encoding a protein that is 
substantially homologous to human TTF-1 protein; (d) a polynucleotide 
capable of hybridizing to any one of polynucleotides (a), (b), or (c); and 
(e) a polynucleotide fragment of any one of polynucleotides (a), (b), (c), 
or (d). 
"Substantially homologous," which can refer both to nucleic acid and amino 
acid sequences, means that a particular subject sequence, for example, a 
mutant sequence, varies from a reference sequence by one or more 
substitutions, deletions, or additions, the net effect of which does not 
result in an adverse functional dissimilarity between reference and 
subject sequences. For purposes of the present invention, sequences having 
greater than 90 percent homology, equivalent biological activity, and 
equivalent expression characteristics are considered substantially 
homologous. For purposes of determining homology, truncation of the mature 
sequence should be disregarded. Sequences having lesser degrees of 
homology, comparable bioactivity, and equivalent expression 
characteristics are considered equivalents. 
In one embodiment, the polynucleotide comprises nucleotides 199 to 569 and 
1,533 to 2,372 of the polynucleotide sequence shown in FIG. 39. In another 
embodiment, the polynucleotide comprises nucleotides 199 to 2,372 of the 
polynucleotide sequence shown in FIG. 39. In yet another embodiment, the 
polynucleotide comprises nucleotides 1 to 2,372 of the polynucleotide 
sequence shown in FIG. 39. In a further embodiment, the polynucleotide 
comprises nucleotides -132 to 3,151 of the sequence shown in FIG. 39. 
The polynucleotides may be employed in the diagnosis of cancers which 
originated in the lung or thyroid. For example, polynucleotide fragments 
of the human TTF-1 protein gene may be produced by PCR. Such 
polynucleotide fragments may be used as diagnostic probes which are 
employed for detecting TTF-1 nucleic acid sequences, such as TTF-1 mRNA, 
in cancer cells. Such detection may be carried out, for example, by 
contacting fixed cancer cells with the polynucleotide probe via in situ 
hybridization, or by isolating the nucleic acids from the cancer cells, 
and contacting such isolated nucleic acids with the polynucleotide probe. 
If the polynucleotide probe binds to nucleic acid sequence(s) of the 
cancer cells, then such cancer has originated in the lung or thyroid, and 
appropriate treatment procedures may be recommended. 
The polynucleotide encoding the human TTF-1 protein also may be placed in 
an appropriate expression vector, which is employed in the transduction of 
cells in vitro, thereby providing for the production in vitro of TTF-1 
protein. Such TTF-1 protein may be used to generate antibodies against 
TTF-1 protein, whereby such antibodies also may be employed as hereinabove 
described for the detection of cancer which originated in the lung or 
thyroid. 
In addition, the promoter region of the polynucleotide encoding human TTF-1 
protein may be placed in an appropriate expression vector in order to 
direct expression of genes encoding lung surfactant proteins, as well as 
other proteins, in lung cells. Such vectors include those hereinabove 
described.

EXAMPLES 
The invention now will be described with respect to the following examples; 
however, the scope of the present invention is not intended to be limited 
thereby. 
Example 1 
Identification of Cis-active Elements Controlling Human Surfactant Protein 
B Gene Expression 
Materials and Methods 
DNase I hypersensitivity-H441 and RAJI cells were disrupted by Dounce 
homogenization in polyamine buffers modified from that of Hewish, et al., 
Biochem. Biophys. Res. Commun., Vol. 52, pgs. 504-510 (1973). 
The use of the polyamine buffer was critical in that DNA purified from 
nuclei that contained calcium exhibit substantial cleavage at the typical 
hypersensitive sites even in the absence of added DNase I. The polyamine 
buffer contained 0.34 M sucrose, 53 mM KCl, 13 mM NaCl, 2 mM EDTA, 0.5 mM 
EGTA, 0.13 mM spermine, 0.5 mM spermidine, 14 mM freshly prepared 
2-mercaptoethanol, 0.1% Triton X-100, 13 mM Tris-HCl, pH 7.4, 3 mM 
MgCl.sub.2, and 1mM freshly prepared phenylmethylsulfonyl fluoride. Nuclei 
were prepared from the homogenates and centrifuged at 2,400.times.g for 30 
minutes over a cushion of 1.2 M sucrose in polyamine buffer. The nuclear 
pellet was washed twice in polyamine buffer without sucrose and detergent 
and resuspended in a DNase I digestion buffer that contained 60 mMKCl, 5 
mM MgCl.sub.2, 0.1 mM EGTA, 0.5 mM dithiothreitol, 5% glycerol, and 15 mM 
Tris-HCl, pH 7.5. Nuclei were resuspended at a concentration of 
1.25.times.10.sup.7 to 3.5.times.10.sup.7 nuclei/ml, and gentle DNase I 
digestions were carried out in a volume of 0.2 ml with 7 units of DNase I 
(Bohringer Mannheim) at 30.degree. C. for 1, 2.5, 5, 10, and 15 minutes. 
Zero time points were not subjected to DNase I. DNA was prepared from 
nuclei treated or untreated with DNase I by the addition of an equal 
volume of a buffer that contained 0.6 M NaCl, 20 mM EDTA, 20 mM Tris-HCl, 
pH 7.5, and 0.5% SDS. The nuclear lysates were digested with 40 .mu.g/ml 
of heat-treated RNase A for 2 hours at 50.degree. C. followed by 300 
.mu.l/ml of proteinase K overnight at 37.degree. C. DNA was purified by 
phenol extraction and ethanol precipitation and quantitated 
spectrophotometrically. DNA samples were digested with HindIII, 
electrophoresed through agarose gels, blotted to Nytran, and hybridized to 
probe radio labeled by means of random primers. The probe was a 1044-bp 
PCR subfragment of the SPB genomic clone PG13-2 (bp 6053-7096) and is 
shown to scale in FIG. 1C. 
Plasmids--The isolation and cloning of the entire SPB gene has been 
reported in Pilot-Matias, et al., DNA, Vol. 8, pgs. 75-86 (1989). Clone 
.lambda. PG13-2 contains the entire SPB gene and more than 2.2 kb of 5' 
flanking sequence (Pilot-Matias, et al., 1989). .lambda. PG13-2 was used 
to clone sequence for all SPB constructions. 
Plasmids pSV40-CAT, pRSV-CAT, and pCMV-.beta.gal have been described in 
Gorman, et al., Mol.Cell.Biol., Vol. 2, pgs. 1044-1051 (1982) and Miller, 
Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, Cold 
Spring Harbor, N.Y., pgs. 352-355 (1972). p2244/436-CAT contains SPB 
genomic sequence from -2244 to +436 in the HindIII site of pSVO-CAT and 
was constructed in three steps. First, the 2.2-kb SalI-KpnI SPB genomic 
fragment (bp -2244 to -4) was subcloned into the corresponding sites of 
pUC19. Second, these sequences were liberated from the polycloning site of 
pUC-19 by digestion with HindIII and EcoRI and introduced into the HindIII 
site of pSVO-CAT with HindIII linkers in a 5' to 3' orientation with 
respect to the CAT gene to give plasmid p2.2-CAT. Sequences downstream of 
the KpnI site (-5 to +436) were amplified from .lambda. PG13-2 using the 
PCR to generate a Kpn I-HindIII- linkered fragment containing a single 
base pair substitution at +15 (A to T). This fragment was cloned into the 
KpnI and downstream HindIII site of p2.2-CAT to give p2244/436-CAT. The 
single base pair change at +15 alters the translation start signal encoded 
in SPB exon I (AUG to DUG) and was necessary to prevent the generation of 
an SPB-CAT fusion protein. (Alam, et al., Biotechniques, Vol. 10, pgs. 
423-425 (1991)). 
5'-Flanking deletions were constructed from p2244/436-CAT by digestion with 
NdeI followed by complete digestion with SacI (p.DELTA.5'-1993), SauI 
(p.DELTA.'-1552), BstEII (p.DELTA.5'-1414). StuI (p.DELTA.5'-900), PpuMI 
(p.DELTA.5'-650), SfiI (p.DELTA.5'-366), or BstXI (p.DELTA.5'-218). 
Recessed 5' or 3' termini were subsequently blunt ended with T4 DNA 
polymerase and plasmids recircularized with T4 DNA ligase. p.DELTA.5'-80 
was constructed using PCR to generate a HindIII-linkered SPB subfragment 
(bp -80 to +436) which was subcloned into the HindIII site of pSVO-CAT. 
Plasmid (pdl (+112/+318)) was constructed by digestion of p2244/436-CAT 
with AvrII and XbaI followed by recircularization. p.DELTA.3'+41 was 
constructed by complete digestion of .DELTA.5'-1993 with HindIII and BspMI 
followed by end filling with T4 DNA polymerase and recircularization. 
p.DELTA.3'+7 contains SPB sequence -2244 to +7 and represents the assembly 
of SalI and PstI subfragments in the HindIII site of pSVO-CAT. Plasmid 
(pdl(+8/+38)) was constructed from p2244/436-CAT by partial digestion of 
PstI followed by recircularization. p218/41 was constructed by digestion 
of p.DELTA.5'-218 with BspMI and HindIII followed by recircularization. 
Following propagation in DH5.alpha. Escherichia coli, the identity of all 
constructions was confirmed by restriction mapping, and PCR subfragment 
sequences were confirmed by dideoxy sequence analysis. 
Cell Culture--Human lung adenocarcinoma cell line NCI-H441 was maintained 
in RPMI medium containing 10% fetal bovine serum. Human lung 
adenocarcinoma cell line A549 and HeLa cells were maintained in Dulbecco's 
modified Eagle's medium containing 10% fetal bovine serum. GM 4671 (RAJI) 
is a human B-lymphoid cell line and was maintained as described in Aronow, 
et al., Genes & Dev., Vol. 3, pgs. 1384-1400 (1989). All cell lines were 
cultured at 37.degree. C. and 5% CO.sub.2. 
Transient Transfection--A mixture of 5 pmol of test plasmid was mixed with 
2.5 pmol of the internal control plasmid pCMV-.beta.gal and coprecipitated 
by the calcium phosphate procedure. Precipitates (1 ml) were added 
directly to the tissue culture medium. Eighteen to 24 hours subsequent to 
transfection the cells were washed and the medium was changed to RPMI with 
10% fetal bovine serum. Cells were harvested by scraping 24 or 48 hours 
later. Assays for .beta.-galactosidase were performed according to Miller, 
1972. (CAT assays were performed as described by Gorman, et al., 
Mol.Cell.Biol., Vol. 2, pgs. 1044-1051 (1982). Chloramphenicol, 
[dichloroacetyl-1, 2-.sup.14 C], and its derivatives were separated by 
thin layer chromatography. The percent acetylation was quantitated using a 
Molecular Dynamics PhosphorImager. To ensure linearity of the assay, data 
were quantitated from CAT assays in which less than 206 conversion had 
occurred. Relative CAT activities were calculated by comparing the 
activities of the promoter-containing plasmids with the activity of 
pSVO-CAT (which produced 0.082% acetylation/unit of .beta.-galactosidase 
activity/h in H441 cells and 0.018% acetylation/unit of 
.beta.-galactosidase activity/h in HeLa cells) within each cell line 
following correction for transfection efficiency. Although transfection 
efficiencies (units .beta.-galactosidase activity/.mu.g protein) and 
absolute CAT conversion varied between experiments (approximately 
2-10-fold), relative CAT activities were similar between experiments. 
DNase I footprinting-HeLa nuclear extracts were made according to Jacob, et 
al., J.Biol.Chem., Vol. 266, pgs. 22537-22544 (1991). H441 extracts were 
made according to Shapiro, et al., DNA, Vol. 7, pgs. 47-55 (1988), with 
modifications as described in Stripp, et al., J.Biol.Chem., Vol. 267, pgs. 
14703-14712 (1992). DNA probes for footprint analysis were prepared by 
using the PCR and .sup.32 P-end-labeled synthetic oligonucleotide primers. 
The SPB genomic clone, 
.lambda. PG13-2, was used as template for the amplification of sequence 
between base pairs -221 and +81. The upstream and downstream primers used 
were (5'-CAGGAACATGGGAGTCTGGG) (SEQ ID NO.: 29) and 
(5'-CAGTGCCTGGGCCACAGAGC), (SEQ ID NO.: 30) respectively. The upstream or 
downstream primer (3 pmol) was .sup.32 P-end-labeled in a 20 .mu.l kinase 
reaction mixture containing 30 pmol of [.gamma..sup.32 P] ATP as 
described. (Maniatis, et al., Molecular Cloning: A Laboratory Manual, 2nd 
ed., pgs. 11.31-11.32, Cold Spring Harbor Laboratory Press (1989). 
Kinase reactions were terminated by incubation at 65.degree. C. for 10 min 
and added directly to a standard 100-.mu.l PCR reaction mixture containing 
3 pmol of unlabeled primer oligonucleotide and 500 ng of template DNA. PCR 
products were isolated using Promega PCR Preps DNA Purification System. 
The DNase I protection assay was performed in a 50-.mu.l reaction. DNA 
binding reactions were carried out in a mixture containing 10 mM Tris, pH 
7.5, 0.5 mM dithiothreitol, 5 mM MgCl.sub.2, 0.1 mM EDTA, 75 mM KCl, 0.2 
mM phenylmethylsufonyl fluoride, and 12% glycerol. Nuclear proteins were 
incubated with 2 .mu.g of poly(dI.dC) competitor DNA for 15 min at 
0.degree. C. prior to the addition of 20,000 counts/min. of labeled DNA 
(about 0.3 ng). After another 60-min incubation at 0.degree. C., the 
samples were set at room temperature and after 5 min. digested with DNase 
I (Promega) for 2 min. The reactions were stopped by the addition of 350 
.mu.l of stop buffer containing 230 mM NaCl, 17 mM EDTA, 1.14% SDS, 11.4 
mM Tris, pH 7.8, and 230 .mu.g/ml proteinase K. DNA was purified by phenol 
extraction and ethanol precipitation. DNA samples were fractionated on 6% 
polyacrylamide, 7 M urea sequencing gels. 
RESULTS 
Identification of DNase I hypersensitive Sites Flanking the SPB 
Promoter--Because many enhancer-like elements and other functional regions 
are associated with perturbations of chromatin structure, DNase I 
hypersensitivity (DH) assays were used to evaluate the SPB gene and 
5'-flanking DNA. A 12.1-kb HindIII fragment was used to map DH sites. This 
fragment contained over 5 kb of 5'-flanking sequence and over 8 kb of 
intragenic sequence extending to HindIII site in intron 10. Autoradiograms 
of the indirectly end-labeled fragments that were generated by DNase I 
treatment of nuclei are shown in FIG. 1, A and B. Nuclei were analyzed 
from a human lung adenocarcinoma cell line (H441), a non-lung cell line 
(RAJI), and human thymus. A total of four hypersensitive sites were 
identified in H441 cell nuclei (Roman numerals, FIG. 1A). These sites, 
designated DNase I-hypersensitive sites I to IV (DHI-DHIV), were located 
proximal to the SPB promoter and within intron eight of the gene. Each 
site was mapped in two separate experiments by comparison of the DNase I 
liberated fragments to known molecular weight standards. The locations of 
these sites are summarized in FIG. 1C. An identical procedure detected no 
DH sites in preparations of RAJI cell nuclei (a non-lung human B-lymphoid 
cell line). (FIG. 1B.). These data suggest that chromatin in H441 cell 
nuclei, but not non-lung cell nuclei, exists in a unique structure which 
is sensitive to DNase I and indicates that important regulatory regions 
may lie in close proximity to the promoter or within the gene. 
Sequences Flanking the SPB Promoter Direct Lung Cell-specific 
Expression--To determine if sequences encompassing DNase I hypersensitive 
sites I and II were associated with functional transcriptional regulatory 
domains, 2.7 kb of sequence (-2244 to +436) was linked to a CAT reporter 
gene. The transcriptional activity of this construction (p2244-436-CAT) in 
the indicated cell lines was determined by transient transfection. Each 
plasmid (5pmol) containing the CAT reporter gene was co-transfected along 
with pCMV.beta.-gal into H441, A549, and HeLa cell lines. CAT activity was 
measured 48 hrs. later and normalized to .beta.-galactosidase activity. 
The activity in each cell line is compared to that of pSV40-CAT. pRSV-CAT 
was employed as an external positive control for CAT activity. Increased 
transcription of the CAT reporter was observed only in H441 cells, where 
an approximate 10-fold increase in expression relative to promoterless 
vector pSVO-CAT was observed (FIG. 2A, lanes 1 and 2). Transfection of 
p2244/436 into A549 cells, a human pulmonary adenocarcinoma cell line that 
does not express SPB, or HeLa cells did not support CAT transcription 
above promoterless vector (FIG. 2, B and C, lanes 1 and 2). This result 
indicated that a human lung adenocarcinoma cell line, H441, was capable of 
expressing chimeric SPB-CAT genes and that the human SPB gene promoter and 
flanking sequences contained within -2244 to +436 was transcriptionally 
active in a cell type-specific manner. 
To determine if sequence encompassing hypersensitive sites III and IV 
contained additional regulatory elements, a genomic subfragment spanning 
intron eight was subcloned into the BamHI site downstream of the CAT 
reporter gene and SPB promoter and flanking sequence (-2244 to +436). The 
transcriptional activity of this construction was similar to p2244/436-CAT 
(data not shown). This result suggested that DHIII and DHIV were not 
associated with a typical enhancer element. 
Deletion Analysis of Sequence Flanking the SPB Promoter--To delineate 
better the cis-acting sequences that regulate SPB transcription in H441 
cells, a series of 5'-flanking deletions of SPB sequence were analyzed in 
transient expression assays. The deletion mutants were constructed as 
hereinabove described, and each 5' deletion mutant had the same 3' end 
point at +436, containing sequence into SPB exon 2. Each plasmid was 
co-transfected with pCMV-.beta.-galactosidase activity. Relative CAT 
activities were calculated by comparing the activities of the SPB promoter 
containing plasmids with those of pSV40-CAT as hereinabove described. A 
summary of the results obtained from transfection of these CAT reporter 
constructs is shown in FIG. 3. As shown in FIG. 3, the lower line shows 
the location of consensus binding site motifs found within the SPB 
promoter region. Each construction was assayed for expression in both H441 
and HeLa cell lines. CAT activity varied in H441 cells with deletion of 
5'-flanking DNA to -218 (p.DELTA.5'-218), but there was no loss of 
activity relative to p2244/436-CAT and no construction expressed above the 
level of pSVO-CAT in HeLa cells. However, deletion of sequence to -80 
(p.DELTA.5'-80) resulted in 82% reduction in reporter activity compared to 
p2244/436-CAT, suggesting that a positive cis-active element was located 
between -218 and -80. 
In order to determine if additional regulatory elements were located 
downstream of the SPB transcription site, a series of 3' introgenic 
deletion mutants was constructed. The extent of each deletion is shown 
relative to p2244/436 by broken lines. Each 3' deletion mutant had the 
same 5' end point at -2244 bp. Each plasmid was co-transfected with 
pCMV-.beta.gal into H441 and HeLa cells, and CAT activity was normalized 
to .beta.-galactosidase activity. Relative CAT activities were calculated 
by comparing the activities of the SPB promoter-containing plasmids with 
those of SV40CAT as hereinabove described. 
A summary of the results obtained from transient expression of these CAT 
reporter constructs in H441 and HeLa cells is shown in FIG. 4. Deletion of 
3'-flanking DNA to +41 (p.DELTA.3'+41) or internal deletion of sequence 
encompassing most of the first intron (pdl(+112/+318)) did not 
significantly alter reporter gene activity. Further deletion of 
3'-flanking DNA to +7 (p.DELTA.3'+7) reduced reporter gene activity by 91% 
compared to p2244/436-CAT. In addition, internal deletion of sequence 
encompassing nucleotides +8 to +38 (pdl(+8/+38)) also reduced 
transcriptional activity by 91%. This result suggests the existence of a 
second positive regulatory element located between +8 and +38. Finally, 
the deletion of both 5'-flanking DNA to -218 and adjacent intragenic DNA 
to +41 (p218/41) demonstrated that a 259-bp promoter fragment was 
sufficient to support a level of cell type-specific CAT expression similar 
to p2244/436-CAT. 
Identification and Cellular Specificity of Nuclear Protein-binding Sites 
within the SPB Promoter--To identify nuclear protein-binding sites within 
the SPB promoter and flanking sequence, DNase I footprinting experiments 
were performed using extract prepared from lung (H441) and non-lung (HeLa) 
cell lines. A 300bp fragment (bp -220 to +80) containing the SPB lung 
cell-specific promoter was subjected to DNase I footprint analysis using 
H441 lung cell and HeLa cell nuclear extracts. The coding (FIG. 5A) and 
non-coding (FIG. 5B) strands of the 300 bp fragment were end labeled and 
incubated in the absence (control, lane 2) or presence of H441 (lane 3) or 
HeLa (lane 4) nuclear extracts before partial digestion with DNase I. 
Standard Maxam and Gilbert purine (A+G) sequencing reactions of the same 
fragments were run in parallel (lane 1). Protected sequences identified 
within H441 nuclear extract are indicated with double lines in FIGS. 5a 
and 5B and labeled SPB-f1 and SPB-f2. Sequences protected by both H441 and 
HeLa nuclear extracts are indicated in FIGS. 5A and 5B with single lines 
and labeled SPB-f3, SPB-f4 and SPB-f5. Arrowheads in FIGS. 5A and 5B 
denote sites hypersensitive to DNase I. 
Five nuclear protein-binding sites were identified using H441 nuclear 
extracts on both the coding and non-coding DNA strands (single and double 
lines, FIG. 5, A and B). In addition, multiple DNase I hypersensitive 
sites, reflected as more intense bands of digestion, were observed between 
and within some of the footprinted regions (arrowheads, FIG. 5, A and B). 
This type of DNase I footprint has been described previously for complex 
promoters and enhancers containing multiple closely spaced cis-active 
elements and may reflect the bending of DNA adjacent to these sites 
(Gottschalk, et al., Mol.Cell.Biol., Vol. 10, pgs. 5486-5495 (1990); Ho, 
et al., Proc.Nat.Acad.Sci., Vol. 86, pgs. 6714-6718 (1989)). 
Two footprinted regions, designated SPB factor 1 (SPB-f1; bp -107 to -93) 
and SPB factor 2 (SPB-f2; bp -90 to -73), were protected only with H441 
cell nuclear extract (double lines, FIG. 5, A and B). The 5'-most binding 
site, SPB-f1, did not contain any previously identified enhancer or 
promoter motif. SPB-f2 contained a sequence motif for hepatocyte nuclear 
factor 5 (HNF-5; TGTTTGT) (SEQ ID NO:31), a transcription factor 
previously described in liver. (Rigaud, et al., Cell, Vol. 67, pgs. 
977-986 (1991); Grange, et al., Nucleic Acids Res., Vol. 17, pgs. 
8695-8709 (1989)). 
Three additional nuclear protein-binding sites were identified in both H441 
and HeLa cell nuclear extracts (single lines, FIG. 5, A and B) and 
designated SPB factor 3 to 5 (SPB-f3 to SPB-f5). SPB-f3 contained a six of 
nine match to the consensus CAAT box. SPB-f4 contained a TATA box and 
Sp1-binding site motif. Notably, SPB-f5 was located entirely within the 
protein coding region of the gene and encompassed a consensus AP1-binding 
site motif (5'-TGAGTCA) (SEQ ID NO: 32). The locations of protected 
sequences and binding site motifs are summarized in FIG. 6. 
As shown in FIG. 6, nuclear protein-binding sites identified within the SPB 
lung cell-specific promoter region are indicated for the coding and 
non-coding DNA strands above and below the nucleotide sequence, 
respectively. Sites detected only with H441 lung cell nuclear extract are 
indicated by double lines and labeled SPB-f1 and SPB-f2. Sites protected 
by both H441 and HeLa nuclear extract are indicated with single lines and 
labeled SPB-f3, SPB-f4 and SPB-f5. The numbers correspond to the limits of 
protection for each binding site. The TATA box, CAAT box, Sp1, and AP1 
consensus binding site motifs are indicated in boldface print. The SPB-f2 
site contains an HNF 5 motif on the non coding strand (5'-TGTTTGT3'-). The 
transcription start site is indicated by an arrow and labeled +1. 
Comparison of the human SPB promoter proximal region to the corresponding 
murine sequence revealed uninterrupted conservation of 11 (TGGAGGGCTCT) 
(SEQ ID NO: 33) and 12 (CAAACACTGAGG) (SEQ ID NO: 34) nucleotides in the 
SPB-f1 and SPB-f2-binding sites, respectively. Much less conservation was 
found in regions protected by both H441 and HeLa cell nuclear extract. 
Only 4 of 16, 6 of 24, and 15 of 19 nucleotides were conserved in the 
SPB-f3-, SPB-f4- and SPB-f5-binding sites, respectively. Within SPB-f4, 
the murine sequence did not contain an Sp1 motif; however, a 7-bp TATA box 
element was conserved. Although an AP1-binding site motif was not 
identified within the murine sequence corresponding to SPB-f5 in exon 1, 
this motif was identified 7 bp downstream of the murine TATA box. Taken 
together, these experiments demonstrate that the SPB promoter proximal 
region contains five nuclear protein-binding sites, two of which bind 
novel lung cell-specific nuclear protein complexes. In particular, with 
the exception of the HNF-5 motif in SPB-f2, the sequence of the DNase I 
footprints specifically protected in H441 cells does not correspond to any 
known promoter or enhancer binding site motif and was conserved between 
the human and murine genes, suggesting that these elements represent novel 
lung cell-specific transcriptional regulatory pathways. 
The above results demonstrate that lung cell-specific transcription of the 
SPB gene is dependent on a 259 bp promoter fragment from base -218 to base 
+41 of the SPB gene. 
In order to identify putative distal regulatory elements, the DNase I 
hypersensitivity assay was exploited. (Gross, et al., Ann.Rev.Biochem., 
Vol. 57, pgs. 159-197 (1988); Eissenberg, et al., Ann.Rev.Genetics, Vol. 
19, pgs. 485-536 (1985)). This method has provided consistent correlation 
between the location of DNA regulatory elements, such as enhancers or 
silencers, and the occurrence of DNase I hypersensitive sites. (Gross, et 
al., 1988; Eissenberg, et al., 1985). The most striking finding in 
examining the DNase I hypersensitivity pattern of the SPB gene and 
5'-flanking region was the cellular specificity of DH sites found close to 
or within the SPB promoter region and the lack of additional 
hypersensitivity within 5 kb of additional upstream sequence. Because 
those enhancers which have been examined are associated with DH sites 
(Gross, et al., 1988; Eissenberg, et al., 1985), this result suggested 
that sequence far upstream of DHI and DHII did not contain characteristic 
enhancer domains. In agreement with this finding, deletion of sequence 
between -2241 and -218 did not significantly alter the maximal 
transcriptional activity of the SPB promoter in transient expression 
assays. Taken together, these data demonstrate that sequences sufficient 
to direct lung cell-specific expression of SPB reside within the proximal 
promoter region. 
DNase I footprint analysis of the human SPB promoter revealed five nuclear 
protein-binding sites between bp -102 and +32. The two 5' -most binding 
sites, SPB-f1 and SPB-f2, interacted with nuclear proteins present only in 
H441 cells, and deletion of these sites resulted in significant reduction 
in the transcriptional activity of the SPB promoter. With the exception of 
an HNF5 motif identified in SPB-f2, the sequence of SPB-f1 and SPB-f2 did 
not contain significant homology to more than 150 functional elements for 
vertebrate genes (Faisst, et al., Nucleic Acids Res., Vol. 20, pgs. 3-26 
(1992)). 
A search of the 5'-flanking regions of genes that are expressed in the 
lung, including human and murine surfactant proteins A and C, and rat 
Clara cell secretory protein, did not reveal elements with significant 
homology to SPB-f1 or SPB-f2. However, it is possible that once important 
bases for binding are identified and/or transcriptional proteins are 
isolated or cloned, binding sites in these or other lung genes will become 
evident. Comparison of the human and murine SPB 5'-flanking sequence 
demonstrated that SPB-f1 and SPB-f2 were evolutionarily conserved in spite 
of sequence divergence outside of this region. The final indication that 
SPB-f1 and SPB-f2 are important to the lung cell specificity of SPB gene 
regulation was the low promoter activity in HeLa cells which lacked SPB-f1 
and SPB-f2 binding activity but contained SPB-f3 to SPB-f5 binding 
activity. 
The finding that SPB promoter region contains two evolutionarily conserved 
and previously undescribed nuclear protein-binding sites and that at least 
one of these sites is not related to any previously described lung 
regulatory region or to other consensus sites, strongly suggests the 
existence of novel lung cell-specific transcription factors. These results 
should facilitate studies designed to elucidate the mechanisms of cell 
type-specific gene expression within the lung. 
Example 2 
Identification of TTF-1 and HNF-3 Binding Sites in SPB Promoter Region 
In Example 1, and in Bohinski, et al., 1993, a region of the human SP-B 
promoter was identified, which was protected specifically by lung cell 
nuclear proteins in DNase I footprinting experiments. Comparison to 
homologous sequences from the mouse SP-B gene promoter revealed two, 14 bp 
blocks of uninterrupted identity within these footprinted regions. (FIG. 
7A). In FIG. 7A, vertical lines indicate identity between the mouse and 
human SP-B promoters, and dashes are gaps inserted for maximal alignment. 
The shaded regions are DNase I footprints determined in the study 
described in Bohinski, et al., 1993. The 55 bp region was used as a probe 
in electrophoretic mobility shift assays, and several specific and 
non-specific complexes were observed. Resolution of these complexes was 
simplified, and non-specific binding was reduced by designing sub-probes 
of this region based on the blocks of conserved sequences and DNase I 
protection. This resulted in two probes, designated SPB-f1 and SPB-f2, as 
shown as thick horizontal lines in FIG. 7A. In order to aid in the 
identification of important complexes, the evolutionary conservation of 
this region, and the idea that the cognate cell-type specific 
transcription factors would also be conserved, were exploited. In this 
example, electrophoretic mobility shift assays were conducted upon nuclear 
extracts from human H441 and mouse MLE-15 lung adenocarcinoma cell lines. 
H441 and MLE-15 nuclear extracts were prepared using a `mini-extract` 
procedure adapted from Schreiber et al., Nucl. Acids Res., Vol. 17, pg. 
6419 1989). All procedures for nuclear extraction were performed on ice 
with ice-cold reagents. Confluent monolayers from 1-4, 10-cm dishes were 
washed twice with 10 ml ice-cold phosphate buffered saline (PBS), 
harvested by scraping into 1 ml PBS and pelleted in a 1.5 ml 
microcentrifuge tube at 3,000 rpm for 5 min. The cell pellet was washed 
once in 1 ml PBS and pelleted as above. The pellet was resuspended in one 
packed cell volume of fresh Buffer A (10 mM HEPES, pH 7.9; 10 mM KC1; 0.1 
mM EDTA; 1.5 mM MgCl.sub.2 ; 0.2% v/v Nonidet P-40; 1 mM Dithiothreitol, 
DTT; 0.5 mM phenylmethylsulfonyl fluoride, PMSF), and cells were lysed 
during a 5 minute incubation with occasional gentle vortexing. A nuclear 
pellet was obtained by microcentrifugation at 3,000 rpm for 5 minutes, and 
the supernatant was the cytoplasmic extract. The nuclear pellet was 
resuspended in one packed nuclear volume of fresh Buffer B (20 mM HEPES, 
pH 7.9; 420 mM NaCl; 0.1 mM EDTA; 1.5 mM MgCl.sub.2 ; 25% v/v glycerol; 1 
mM DTT; 0.5 mM PMSF) and nuclei were extracted during a 10 minute 
incubation with occasional gentle vortexing. Extracted nuclei were 
pelleted in a microcentrifuge at 14,000 rpm for 10 minutes. The 
supernatant was recovered and typically contained 5.0-10.0 .mu.g 
.mu.l.sup.-1 of extracted nuclear protein. Nuclear extracts were stored at 
-80.degree. C. without loss of activity for at least six months. 
For the electrophoretic mobility shift assays, oligonucleotides were 
annealed at 10 .mu.M in 100 .mu.l Buffer M (10 mM Tris pH 7.5; 10 mM 
MgCl.sub.2 ; and 50 mM NaCl) by placing the mixture in a preheated 
95.degree. C. dry block which was then slowly cooled to room temperature. 
A.sub.260 was determined and dilutions of this mixture were made in TE (10 
mM Tris pH 8.0; 1 mM EDTA) and used directly in EMSA as unlabeled 
competitor DNA. For use as probe in EMSA 20 .mu.l of the annealed mixture 
was gel purified using a 4% BIOGEL and MERmaid kit as specified by the 
manufacturer (BIO 101). A.sub.260 was determined and 1.5 pmol of annealed 
and gel-purified oligonucleotide were end-labeled using [.gamma..sup.-32 
P]ATP and T4 polynucleotide kinase. End-labeled probe was purified from 
unincorporated [.gamma..sup.-32 P]ATP using a Pharmacia Nick Column and 
recovered in 400 .mu.l TE for an activity of approximately 25,000 
dpm/.mu.l.sup.-1. 
The electrophoretic mobility shift assay (EMSA) was adapted from 
Hennighausen and Lubon, Meth. Enzymol., Vol. 152, pgs. 727-735 (1987). 
Briefly, nuclear extract (1-2 .mu.l) and, when indicated, unlabeled 
oligonucleotide competitor DNA were preincubated in 20 .mu.l Buffer C (12 
mM HEPES, pH 7.9; 4 mM Tris-Cl pH 7.9; 25 mM KC1; 5 mM MgCl.sub.2 ; 1 mM 
EDTA; 1 mM DTT; 50 ng .mu.l.sup.-1 poly[d(I-C)], Boehringer Mannheim; 0.2 
mM fresh PMSF) for 10 minutes on ice. Probe (100,000 dpm) was added and 
incubated an additional 20 minutes on ice. For antibody supershift and 
interference assays, 1 .mu.l of antibody was added after the addition of 
probe and incubated an additional 20 minutes on ice. TTF-1 antibody is 
described in Lazzaro et al., Development, Vol. 113, pgs. 1093-1104 (1991). 
HNF-3.alpha., .beta., and .gamma. antibodies were kindly provided by Dr. 
J. E. Darnell, Jr. (Lai et al., Genes and Devel., Vol. 5, pgs. 416-427 
(1991)). Recombinant, bacterially expressed TTF-1 homeodomain protein 
(TTF-1 HD) is described in Guazzi et al., EMBO J., Vol. 9, pgs. 3631-3639 
(1990). Assays were performed using 1 .mu.l TTF-1 HD in place of nuclear 
extract. Bound and free probe were resolved using non-denaturing 
polyacrylamide gel electrophoresis. 5% gels (acrylamide:bisacrylamide, 
29:1, 0.5.times.TBE (44.5 mM Tris; 44.5 mM Borate; 1 mM EDTA; pH 8.3); 
2.5% v/v glycerol; 1.5 mm thick) were run in 0.5.times.TBE running buffer 
at constant current (30 mA) for approximately 90 minutes. Gels were 
blotted to Whatman 3MM paper, dried under vacuum and exposed to X-ray film 
for 1-3 hours at -80.degree. C. with an intensifying screen. 
Nuclear extracts from both the H441 and MLE-15 cell lines formed two 
complexes of identical electrophoretic mobility with SPB-f1 (FIG. 7B, 
lanes 1 and 2, A and B arrows) and, similarly, one complex of identical 
electrophoretic mobility with SPB-f2 (FIG. 7B, lanes 3 and 4, C arrow). 
Complex D (FIG. 7B, lane 3, D arrow) resolved from Complex C by extended 
electrophoresis, and only appeared using MLE-15 nuclear extracts. A 
complex of low abundance and high mobility, apparent with H441 nuclear 
extract and SPB-f1 (FIG. 7B, lane 2), was not reproducible under these 
conditions. In order to identify Complex D as well as the conserved 
Complexes A, B, and C, MLE-15 nuclear extract was used for further study. 
The binding specificity of these complexes was determined by the addition 
of unlabeled competitor oligonucleotides, referred to in FIGS. 7C and 7D 
as Comp. Each competitor was added in the molar excesses shown in FIGS. 7C 
and 7D. This resulted in efficient competition for complexes A, B, C, and 
D by an excess of self (FIGS. 7C and 7D, lanes 2 and 3), the mouse 
homologue of self (FIGS. 7C and 7D, lanes 4 and 5), but not the respective 
adjacent binding site (FIGS. 7C and 7D, lanes 6 and 7). For SPB-f2, the 
human sequence appeared to be a better competitor than the mouse, but both 
were significantly more efficient competitors than the adjacent binding 
site SPB-f1. Because SPB-f1 and SPB-f2 did not cross compete in these 
assays, it was concluded that at least two distinct and evolutionarily 
conserved nuclear factors specifically bound this region. 
SPB-f2 contained a TGT3 motif (TGTTTGC) (SEQ ID NO: 35) that occurs in the 
regulatory elements of diverse liver-specific genes (Jackson, et al., Mol. 
Cell Biol., Vol. 13, pgs. 2401-2410 (1993)). Because of its apparent 
novelty, this motif also was termed HNF-5 to distinguish it from motifs 
recognized by other liver transcription factors, including HNF-3 (Grange, 
et al., Nucleic Acids Research, Vol. 19, pgs. 131-139 (1990); Rigaud, et 
al., Cell, Vol. 67, pgs. 977-986 (1990)). This motif binds HNF-3 proteins 
(Drewes, et al., Nucleic Acids Research, Vol. 19, pgs. 6383-6389 (1991); 
Jackson, et al., 1993; Nitsch, et al., Genes & Devel., Vol. 7, pgs. 
308-319 (1993); Pani, et al., Mol. Cell. Biol., Vol. 12, pgs. 552-562 
(1993)). SPB-f2 was not clearly related to the HNF-3 motif identified in 
the transthyretin (TTR) and .alpha.-1-antitrypsin liver-specific 
regulatory regions (FIG. 8A, Costa, et al., Nucleic Acids Research, Vol. 
19, pgs. 4139-4145 (1989)). As shown in FIG. 8A, nucleotides that match 
SPB-f2 are shaded in the TGT3 and TTR-S oligos. TGT3 is oligo S4 (Grange, 
et al., 1990) from the tyrosine aminotransferase gene enhancer. TTR-S is 
oligo TTR-S from the TTR gene promoter. (Costa, et al., 1989). mTGT3 
contains a 2 bp mutation that eliminates specific binding of HNF-3 and is 
the same as oligo S4 mut. (Grange, et al., 1990.) oligonucleotides 
representative of each HNF-3 motif were employed as unlabeled competitors 
in an electrophoretic mobility shift assay, and efficient cross 
competition between the motifs was found. As shown in FIG. 8B, unlabeled 
competitors were added to the EMSA assays at a 1,000-fold molar excess as 
compared to probe. A TGT3 site from the tyrosine aminotransferase gene 
enhancer (Grange, et al., 1990) or the strong HNF-3 site from the TTR gene 
promoter, TTR-S (Costa, et al., 1989), were efficient competitors for 
complexes C and D (FIG. 8B). A mutant TGT3 motif (mTGT3) which does not 
bind HNF-3 (Grange, et al., 1990) did not compete for complex C or D (FIG. 
8B). Antisera to electrophoretic mobility shift assay reactions specific 
for each HNF-3 protein (anti-HNF-3.alpha., .beta., and .gamma.) (Lai, et 
al., 1991), were added, and the binding of both HNF-3.alpha.and 
HNF-3.beta. to SPB-f2 was shown using MLE-15 nuclear extracts. H441 
nuclear proteins formed only Complex C. The protein was determined to be 
HNF-3.alpha.. Anti-HNF-3.alpha. and anti-HNF-3.beta. significantly 
interfered with the formation of Complex C and Complex D, respectively, 
and formed only minor supershifted complexes of lower mobility. (FIG. 8C, 
.alpha. and .beta. asterisks). The identification of the lowest mobility 
complex as HNF-3.alpha. was consistent with the relative mobilities of 
HNF-3 proteins in liver cells where HNF-3.beta. complexes migrate only 
slightly faster than HNF-3.alpha. and the two complexes appear as a single 
broad band in an electrophoretic mobility shift assay. (Lai, et al., 
1991). Simultaneous addition of both anti-HNF-3.alpha. and 
anti-HNF-3.beta. eliminated all major complex formation with SPB-f2, and 
indicated that other proteins did not independently bind this region (FIG. 
8C, lane 5). These results were due to specific behavior of 
anti-HNF-3.alpha. and anti-HNF-3.beta. because they did not significantly 
affect Complex A and Complex B (FIG. 8C, lanes 6 and 7), and supported the 
idea that factors bound to SPB-f1 were distinct. In addition, 
anti-HNF-3.gamma. did not affect specifically major complex formation 
(FIG. 8, lane 4), consistent with its lack of expression in the lung (Lai, 
et al., 1991). These observations were supported using Northern blot 
analysis, and expression of HNF-3.alpha. and HNF-3.beta. was detected in 
MLE-15 cells, and only HNF-3.alpha. in H441 cells. 
An informative cis-active motif was not apparent in SPB-f1. In 
electrophoretic mobility shift assay (EMSA), Complex A appeared at high 
nuclear protein concentration, and was eliminated before Complex B by 
unlabeled self-competitor (data not shown). This suggested that two 
factors might bind SPB-f1 independently to form a trimeric protein-DNA 
complex. This hypothesis was tested by using 5' (5'f1) or 3' (3'f1) 
sub-fragments of SPB-f1 as competitors and probes in EMSA (FIGS. 9A and 
9B). The sub-fragments were extended 4 bp beyond SPB-f1 in this region to 
prevent the oligonucleotide from being too small for EMSA. Unlabeled 
competitors were added to the EMSA reactions at a 100-fold molar excess 
compared to probe. The 5' and 3' sub-fragments of SPB-f1 were specific and 
equivalent competitors for Complex A and Complex B, but slightly less 
efficient than the parent fragment (FIG. 9B, lanes 1-5), and this agreed 
with the idea that each sub-fragment had only half the number of binding 
sites as compared with the parent. When labeled and used as a probe, the 
two sub-fragments formed complexes of identical mobility as compared to 
each other, but different from either Complex A or Complex B (FIG. 9B, 
lanes 6 and 7). This could be due to the binding of a factor which induces 
a DNA bend closed to the center of each sub-fragment, but closer to each 
end of the parent fragment. When such a factor binds to the center of a 
DNA molecule, its migration is more retarded in polyacrylamide matrices 
than when bound to the end of the DNA molecule (Wu, Nature, Vol. 308, pgs. 
509-513, (1984)). The idea that the same factor bound to each end of 
SPB-f1 prompted a detailed self to self comparison of these sequences. 
Maximal alignment of 5'f1 and 3'f1 showed less than 50% identity, but 
revealed a short, conserved inverted palindrome motif, CTNNAG (FIG. 10A). 
The first two lines of FIG. 10A show this maximal alignment. The two 
CTNNAG motifs were spaced exactly 10 base pairs from their center point 
within SPB-f1 and were part of larger but distinct inverted palindromes 
(FIG. 10A). A consensus (also known as SPB-f1 con) from this alignment was 
determined (FIG. 10A), and was compared manually to a list of cis-active 
motifs for vertebrate-encoded transcription factors (Faisst, et al., 
Nucleic Acids Res., Vol. 20, pgs. 3-26 (1992)) with emphasis on the CTNNAG 
motif. The SPB-f1 con sequence is shown in line 3 of FIG. 10A, and 
compared with the reported TTF-1 consensus, shown in line 4 of FIG. 10A. 
The sequence of the strong TTF-1 binding site from the thyroglobulin gene 
promoter, oligo C, is shown in line 5 of FIG. 10A. FIG. 10B depicts the 
organization of CTNNAG motifs (shaded) within SPB-f1. Each motif is 
embedded in a larger inverted palindrome indicated above and below the 
sequence by opposing arrows, and labeled I and II. The motifs are 
separated by exactly 10 bp from their centers of dyad symmetry. Several 
motifs shared the CTNNAG core; however, the limited identity to the TTF-1 
binding site was found to be the most attractive because this factor is 
expressed in the developing lung epithelium (Lazzaro, et al., 1991). Using 
the same strategy as hereinabove described for the identification of the 
HNF-3 binding sites, it was found that a high affinity binding site for 
TTF-1 from the thyroid-specific thyroglobulin promoter, oligo C 
(Civitareale, et al., EMBO J., Vol. 8, pgs. 2537-2542 (1989)) was a more 
efficient competitor for Complex A and Complex B than self. (FIG. 10C, 
lanes 2-4). In the experiment in which the results are shown in FIG. 10C, 
unlabeled competitors were added at a 100-fold molar excess as compared to 
probe. When used as a probe, oligo C formed a complex of identical 
mobility to Complex B, consistent with the single TTF-1 binding site in 
this oligo (FIG. 10C, lanes 1 and 2). Oligo C, however, diverges from the 
consensus and does not contain a perfect CTNNAG motif. (FIG. 10A). This 
favored recognition of the defined TTF-1 binding site in oligo C as 
opposed to circumstantial recognition of a CTNNAG motif. Affinity 
purified, polyclonal antisera to TTF-1 in EMSA reactions (anti TTF-1, 
Lazzaro, et al., 1991) was employed, and binding of TTF-1 to two 
independent sites in SPB-f1 was shown. Addition of anti-TTF-1 to EMSA 
reactions containing either MLE-15 or H441 nuclear proteins resulted in 
the elimination of Complex A and Complex B, and the formation of a lower 
mobility complex of similar abundance (FIG. 10D, lanes 1 and 2, and data 
not shown). This reaction was specific because anti-TTF-1 did not alter 
HNF-3 and SPB-f2 complex formation (FIG. 10D, lanes 3 and 4). For lanes 
5-8 of FIG. 10D, unlabeled competitors were added to the EMSA reaction at 
a 100-fold molar excess as compared to probe. Further, a recombinant 
fragment of TTF-1 containing the homeodomain (TTF-1, HD, Guazzi, et al., 
1990) specifically bound to SPB-f1 and formed a two-banded pattern (A' and 
B' in FIG. 10D, lanes 5-8). Complex A' formed at higher protein 
concentrations and depended on the integrity of both CTNNAG motifs in 
SPB-f1 (FIG. 11C). As will be explained hereinbelow, disruption of either 
CTNNAG core motif resulted in complete loss of Complex A' and a reduction 
in Complex B' (FIG. 11C, lanes 2 and 3). Disruption of both sites 
completely eliminated formation of a specific complex (FIG. 11C, lane 4). 
The binding of recombinant TTF-1 HD to either site alone formed complexes 
of slightly different mobility which were evident only when the other site 
was mutated. 
FIG. 11A depicts the relative location and identity of TTF-1 and HNF-3 
binding sites which have been identified. Line one in FIG. 11A shows the 
locations of TTF-1 and HNF-3 binding sites identified in the SP-B 
promoter. Mutations at each site then were constructed, and binding was 
shown to be dependent upon a specific sequence because a 2 bp mutation at 
each site severely impaired factor binding in EMSA experiments. The shaded 
nucleotides in line two indicate the 2 bp mutations that were made at each 
binding site. 
Plasmids containing mutated TTF-1 and HNF-3 binding sites were constructed 
as follows: 
The human surfactant protein B gene promoter (bp -218 to 44) was isolated 
from p2244/436 (Bohinski et al., 1993) (FIG. 12) using PCR and linker 
primers to create 5' HindIII and 3' SalI sites. The product was digested 
with HindIII and SalI and cloned into the respective sites of M13mp-18 
(Gibco-BRL, catalog no. 18227-017) and used as template for site directed 
mutagenesis performed by the method of Kunkel, Proc. Nat. Acad. Sci., USA, 
Vol. 82, pgs. 488-492 (1985). The wild type and mutated promoters were 
isolated from M13 replicative form by HindIII and SalI digestion and 
cloned into the respective sites of pBLCAT6 (Boshart, et al., Gene, Vol. 
110, pgs. 129-130 (1992)). (FIG. 13) These SPB promoter-CAT plasmids were 
designated p218/41-WT (FIG. 14), -5T, -3T, -TT, -H, or -TTH, and 
identities were confirmed dideoxy sequencing of double stranded templates. 
Plasmid p128/41-WT contains no mutations in the human surfactant protein B 
gene promoter region. Plasmid p218/41-5T contains a mutation in the 5' 
TTF-1 binding site in the region from bp-118 to bp-64 of the human 
surfactant protein B gene promoter region. Plasmid p218/41-3T contains a 
mutation in the 3' TTF-1 binding site in the region from bp-118 to bp-64 
of the human surfactant protein B gene promoter region. Plasmid p218/41-TT 
contains mutations in the 5' TTF-1 binding site and in the 3' TTF-1 
binding site in the region from bp-118 to bp-64 of the human surfactant 
protein B gene promoter region. Plasmid p218-41-H contains a mutation in 
the HNF-3 binding site in the region from bp-118 to bp-64 of the human 
surfactant protein B gene promoter region. Plasmid p218/41-TTH contains 
mutations in the 5' TTF-1 binding site, the 3' TTF-1 binding site, and the 
HNF-3 binding site in the region from bp-118 to bp-64 of the human 
surfactant protein B gene promoter region. The 5' deletion mutant 
p.DELTA.-80 contains human SPB (bp -80 to 41) in the HindIII and SalI 
sites of pBLCAT6 (FIG. 13) and was made using PCR and linker primers as 
above. The rat CCSP gene promoter (bp -2338 to 49) was cloned into the 
polylinker of pBLCAT6 (FIG. 13) as described in Stripp, et al., Genomics, 
Vol. 20, pgs. 27-35 (1994) and was kindly provided by Dr. B. R. Stripp. 
The mouse SPC gene promoter (bp -4680 to 18) was isolated as an XbaI and 
HpaII fragment, digested with nuclease Bal31 at its 3' end, repaired with 
T4 DNA polymerase to bp 18, and cloned as an XbaI and 3' XhoI-linked 
fragment into the respective sites of pBLCAT6 (FIG. 13). pBLCATS contains 
the thymidine kinase promoter (bp -105 to 51) (Boshart, et al., 1992). 
TTR-CAT contains the mouse transthyretin promoter (bp -202 to 9) and was 
kindly provided by Dr. J. E. Darnell, Jr. (Lai, et al., 1991). 
NCI-H441-4 (H441) and MLE-15 cells (used in nuclear extract procedure) were 
maintained exactly as described in O'Reilly, et al., 1988 and Wikenheiser, 
et al., Proc. Nat. Acad. Sci., Vol. 901, pgs. 11029-11033 (1993). HeLa 
cells were maintained in Dulbecco's Modified Eagle Medium containing 10% 
heat inactivated fetal bovine serum. The day before transfection confluent 
monolayers were split (1:5-1:8 for H441 cells; 1:20 for HeLa cells) into 
10-cm dishes. Four hours before transfection cells were switched to 
transfection medium (Dulbecco's Modified Eagle Medium containing 10% heat 
inactivated fetal bovine serum and 1% penicillin-streptomycin, Gibco BRL). 
Transfections were performed using the calcium phosphate coprecipitation 
method essentially as described (Rosenthal, Meth. Enzymol., Vol. 152, pgs. 
704-720 (1987)) except glycerol shock was not used. For the analysis of 
point mutants in H441 cells precipitates were prepared using 5.0 pmol of 
promoter-CAT fusion plasmid and 2.5 pmol of the internal control plasmid, 
pCMV-.beta.gal, (MacGregor, et al., Nucleic Acids Res., Vol. 6, pg. 2365 
(1989)) per 10-cm dish. Precipitates were added dropwise to the medium 
covering the cells. The cells were incubated with precipitate for 14-18 
hours, washed once with calcium and magnesium free Hanks' Balanced Salt 
Solution, returned to maintenance medium and cultured for an additional 24 
hours. Cells were harvested and freeze-thaw lysates were prepared in 100 
.mu.l of 0.25M Tris, pH7.8, and aliquots assayed for CAT activity and 
.beta.-galactosidase activity as described in Rosenthal (1987) and 
MacGregor, et al., Methods in Molecular Biology, Murray, ed., Vol. 7, pgs. 
217-235, Humana Press, Clifton, N.J. (1991). To correct for variations in 
transfection efficiency, lysates were normalized for .beta.-galactosidase 
activity that CAT enzyme assays contained equivalent amounts of 
.beta.-galactosidase activity. Thin layer chromatograms of .sup.14 
C-chloramphenicol and its acetylated derivatives were quantitated using a 
Molecular Dynamics Phosphor Imager. 
The results of the transfection experiments were as follows. The mutated 
version of SPB-f2 (H) did not compete for or bind HNF-3 proteins (FIG. 
11D), and, as discussed above, TTF-1 binding depended upon the integrity 
of the CTNNAG motif. (FIG. 11C). For the experiments in which the results 
are shown in FIG. 11C, 1 .mu.l of TTF-1 HD was used in place of nuclear 
extract, and incubated with the wild type SPB-f1 probe (f1) or, with one 
of the mutant probes 5T, 3T, or TT in EMSA assays. For the experiments in 
which the results are shown in FIG. 11D, the wild type SPB-f2 probe was 
compared to the mutant probe H in an EMSA assay using MLE-15 nuclear 
extract. Unlabeled competitors were added at a 1,000-fold molar excess 
compared to probe. In order to determine if these sites were 
transcriptionally active, site-directed mutagenesis was used to construct 
these binding site mutations in the SPB gene promoter. As hereinabove 
described, the wild type (WT) and mutant promoters were linked to a CAT 
reporter gene and assayed for transcriptional activity in H441 and HeLa 
cells. (FIG. 11B). For the wild type promoter, CAT activity equals 1.00. 
The results shown are average values from 3 independent experiments where 
the standard error of the mean was less than 10%. All mutations resulted 
in a statistically significant reduction in CAT activity in H441 cells, 
and no mutation affected activity in HeLa cells, thus demonstrating the 
restricted cellular activity of factors bound to this region. Mutation of 
the 5' TTF-1 binding site (5T) was less dramatic than mutation of the 3' 
TTF-1 binding site (3T), and mutation of both TTF-1 sites (TT) was no 
different than for the 3T mutation, suggesting that the 5' site depended 
on the 3' site for activity. Mutation of all three binding sites (TTH) 
resulted in an activity that was not different from gross deletion of all 
sequences upstream of -80 (.DELTA.-80). This indicated that no other sites 
were present between -218 and -80 or that no other site in this region 
could affect SPB promoter function in the absence of the defined TTF-1 and 
HNF-3 sites. Although each site demonstrated transcriptional activity, 
complementary HNF-3 (H) and TTF-1 (TT) mutations accounted for only 41% of 
wild type activity. Thus, it is concluded that TTF-1 and HNF-3 proteins 
synergistically activate SPB promoter function from this region. 
It was then reasoned that TTF-1 would function as a binding site depending 
transactivator of SPB and other target promoters, and the SPB promoter and 
binding site mutants were employed to develop an assay for the DNA-binding 
and transcriptional activating function of TTF-1. HeLa cells were 
transfected with plasmids containing wild-type or mutant SPB promoters, 
and either the empty vector pRc/CMV (Invitrogen) or an vector containing 
the entire TTF-1 open reading frame (pCMV-TTF-1) (Francis-Lang, et al., 
Mol. Cell Biol., Vol. 12, pgs. 576-588 (1992)). For the TTF-1 
transactivation experiments in HeLa cells each 10 cm dish was treated with 
a precipitate prepared using 15.0 .mu.g promoter-CAT fusion plasmid, 2.0 
.mu.g pCMV-.beta.gal, 7.5 .mu.g pUC19, and 0.5 .mu.g of either the empty 
vector pRc/CMV (Invitrogen), or the pCMV-TTF-1 vector containing the 
entire TTF-1 open reading frame. Precipitates were added dropwise to the 
medium covering the cells. Cells were incubated with precipitate for 14-18 
hours, washed once with calcium and magnesium from Hanks' Balanced Salt 
Solution, returned to maintenance medium, and cultured for an additional 
48 hours. Cells were harvested and freeze-thaw lysates were prepared in 
100 .mu.l 0.25 M Tris, pH 7.8, and aliquots were assayed for CAT and 
.beta.-galactosidase activity essentially as described in Rosenthal (1987) 
and MacGregor, et al. (1991). In order to correct for variations in 
transfection efficiency, lysates were normalized for .beta.-galactosidase 
activity so that CAT enzyme assays contained equivalent amounts of 
.beta.-galactosidase activity. Thin layer chromatograms of .sup.14 
C-chloramphenicol and its acetylated derivatives were quantitated using a 
Molecular Dynamics Phosphor Imager. For the experiments in which the 
results are shown in FIG. 15A, the wild type (WT), TT, or H SPB promoter 
constructs were co-transfected transiently with the internal control 
plasmid pCMV.beta.-gal and either the empty vector (-), or vector 
containing the full length TTF-1 cDNA (+), into the HeLa cell line. Each 
(+) or (-) determination is representative of three independent 
experiments that were normalized from .beta.-galactosidase. For the 
experiments in which the results are shown in FIG. 15B, CCSP, SPC, TTR, or 
TK promoter constructs were co-transfected (-) or (+) into the HeLa cell 
line as hereinabove described, and each determination is representative of 
three independent experiments. As shown in FIG. 15A, TTF-1 dramatically 
increased activity from the wild-type SPB promoter (FIG. 15A, lanes 1 and 
2), but had no effect on the TTF-1 mutant promoter (FIG. 15A, lanes 3 and 
4). Co-transfected TTF-1 also strongly activated the HNF-3 mutant promoter 
(FIG. 15A, lanes 5 and 6). Because TTF-1 transactivation was dependent 
strictly on the integrity of TTF-1 binding sites, these results 
demonstrated further a direct effect of TTF-1 on SPB promoter activity. 
This system then was employed to demonstrate the transcriptional response 
of other lung-specific promoters to TTF-1. TTF-1 dramatically increased 
the activity of the lung-specific CCSP and SPC gene promoters, but had no 
effect on the liver-specific TTR or the constitutive thymidine kinase (TK) 
gene promoters (FIG. 15B). 
Example 3 
Construction of an Adenoviral Vector for Lung Surfactant Gene Therapy Which 
Expresses the Surfactant Protein B Gene and Utilizes the Cognate 
Surfactant Protein B Gene Promoter 
The purpose of developing this vector for gene therapy for human surfactant 
protein deficiency states is to improve upon existing adenoviral vectors 
including DNA encoding human surfactant protein B. One current vector, 
AvSPB1 (disclosed in U.S. patent application Ser. No. 08/044,406, filed 
Apr. 8, 1993, now abandoned, incorporated herein by reference), expresses 
human surfactant protein B under control of the Rous Sarcoma Virus (RSV) 
long terminal repeat. This expression, however, is constitutive and not 
regulated by the usual transcriptional signals which modulate the 
endogenous SP-B gene in health and disease. The new vector Av1SPB2 (FIG. 
22), the construction of which is described hereinbelow, is designed to 
express the human surfactant protein B gene under the control of its 
cognate human surfactant protein B gene promoter. This will allow for lung 
specific gene expression, and further, will allow for correct regulation 
of the gene after transfer into the patient's lung cells. 
A similar vector, Av1SPB3 (FIG. 22), the construction of which is described 
hereinbelow, is designed to express the human surfactant protein B gene 
under the control of the murine surfactant protein B gene promoter. 
Construction of this vector allows evaluations to be carried out in a 
murine model to verify the tissue-specificity in an animal model prior to 
evaluations of the cognate human promoter-structural SPB gene in human 
clinical trials of SPB deficiency states. 
A. Construction of pAVS6 
The adenoviral construction shuttle plasmid pAvS6 was constructed in 
several steps using standard cloning techniques including polymerase chain 
reaction based cloning techniques. First, the 2913 bp BglII, HindIII 
fragment was removed from Ad-dl327 and inserted as a blunt fragment into 
the XhoI site of pBluescrpt II KS-(Stratagene, La Jolla, Calif.) (FIG. 
16). 
Ad-dl327 (Thimmappaya, et al., Cell, Vol. 31, pg. 543 (1983)) is identical 
to adenovirus 5 except that an XbaI fragment including bases 28591 to 
30474 (or map units 78.5 to 84.7) of the Adenovirus 5 genome, and which is 
located in the E3 region, has been deleted. The complete Adenovirus 5 
genome is registered as Genbank accession #M73260, incorporated herein by 
reference, and the virus is available from the American Type Culture 
Collection, Rockville, Md., U.S.A. under accession number VR-5. 
Ad-dl327 was constructed by routine methods from Adenovirus 5 (Ad5). The 
method is outlined briefly as follows and previously described by Jones 
and Shenk, Cell 13:181-188 (1978). AdS DNA is isolated by proteolytic 
digestion of the virion and partially cleaved with Xba 1 restriction 
endonuclease. The Xba 1 fragments are then reassembled by ligation as a 
mixture of fragments. This results in some ligated genomes with a sequence 
similar to Ad5, except excluding sequences 28593 bp to 30470 bp. This DNA 
is then transfected into suitable cells (e.g. KB cells, HeLa cells, 293 
cells) and overlaid with soft agar to allow plaque formation. Individual 
plaques are then isolated, amplified, and screened for the absence of the 
1878 bp E3 region Xba 1 fragment. 
The orientation of this fragment was such that the BglII site was nearest 
the T7 RNA polymerase site of pBluescript II KS. This plasmid was 
designated pHR. (FIG. 16). 
Second, the ITR, encapsidation signal, Rous Sarcoma Virus promoter, the 
adenoviral tripartite leader (TPL) sequence and linking sequences were 
assembled as a block using PCR amplification (FIG. 17). The ITR and 
encapsidation signal (sequences 1-392 of Ad-dl327 [identical to sequences 
from Ad5, Genbank accession #M73260] incorporated herein by reference) 
were amplified (amplification 1) together from Ad-dl327 using primers 
containing NotI or AscI restriction sites. The Rous Sarcoma Virus LTR 
promoter was amplified (amplification 2) from the plasmid pRC/RSV 
(sequences 209 to 605; Invitrogen, San Diego, Calif.) using primers 
containing an AscI site and an SfiI site. DNA products from amplifications 
1 and 2 were joined using the "overlap" PCR method (amplification 3) 
(Horton, et al., BioTechniques, 8:528-535 (1990)) with only the NotI 
primer and the SfiI primer. Complementarity between the AscI containing 
end of each initial DNA amplification product from reactions 1 and 2 
allowed joining of these two pieces during amplification. Next the TPL was 
amplified (amplification 4) (sequences 6049 to 9730 of Ad-dl327 [identical 
to similar sequences from Ad5, Genbank accession #M73260]) from cDNA made 
from mRNA isolated from 293 cells (ATCC Accession No. CRL 1573) infected 
for 16 hrs. with Ad-dl327 using primers containing SfiI and XbaI sites 
respectively. DNA fragments from amplification reactions 3 and 4 were then 
joined using PCR (amplification 5) with the NotI and XbaI primers, thus 
creating the complete gene block. 
Third, the ITR-encapsidation signal-TPL fragment was then purified, cleaved 
with NotI and XbaI and inserted into the NotI, XbaI cleaved pHR plasmid. 
This plasmid was designated pAvS6A.sup.- and the orientation was such 
that the NotI site of the fragment was next to the T7 RNA polymerase site 
(FIG. 18). 
Fourth, the SV40 early polyA signal was removed from SV40 DNA as an 
HpaI-BamHI fragment, treated with T4 DNA polymerase and inserted into the 
SalI site of the plasmid pAvS6A- (FIG. 18) to create pAvS6 (FIGS. 18 and 
19). 
The vectors Av1SPB2 and Av1SPB3 then are constructed as follows. First, the 
region of SP-B promoter which contains the essential SP-B regulatory 
elements (bp-439 to bp +41; Bohinski, et al., 1993) are cloned into the 
promoter position in pAvS6 (FIG. 19) in place of the RSV promoter which is 
first removed, by standard PCR cloning methods. The murine SPB promoter 
was cloned by using the following 5' and 3' primers: 
Murine SPB5':5'-TGGACAGGCGCGCC CGGCACTTACCC TGCGTCAAGAGCCAGGAAGG-3' 
(SEQ ID NO.:36) 
AscI 
- Murine SPB3':5'-CGTCATGGCCATATGGGCC TAGCCACTGCAG TAGGTGCGACTTGGCCATGG 
-3' (SEQ ID NO.:37) 
SfiI 
The human SPB promoter was cloned by using the following 5' and 3' primers: 
Human SPB5':5'-TGGACAGGCGCGCC CAGGGCTTGCCCTGG GTTAAGAGCCAGGCAGG-3' 
(SEQ ID NO.:38) 
AscI 
- Human SPB3':5'-CGTCATGGCCATATGGGCC CAGCCACTGCAG CAGGTGTGACTCAGCCATGG- 
3' (SEQ ID NO.:39) 
SfiI 
Second, after PCR amplification of the correct region from the SPB promoter 
containing plasmid (PMSPB (murine) (FIG. 20); pHSPB (human) (FIG. 21)), 
the PCR product is cloned into a minimal promoter expression plasmid 
containing the critical left end viral elements used in the adenovirus 
vector construction shuttle plasmid pAvS6. (FIG. 19). 
The resulting plasmid vector contains the following sequential elements: 
the Ad5 left inverted terminal repeat (ITR), the encapsidation signal 
sequence, the SPB promoter element (from -439 bp to +44 bp for the human 
promoter, or from -382 bp to +41 bp for the murine promoter) followed by 
the remainder of pAvS6 (FIG. 19). 
Third, this plasmid is linearized at the EcoRV site, the human SP-B gene is 
inserted so that the 5' end of the coding strand is closest to the 
promoter element. This plasmid then is linearized and co-transfected with 
the large fragment of Ad dl327 in 293 cells to generate the final 
adenoviral vector shown in FIG. 22. 
The SP-B-adenoviral vector is formulated for aerosol instillation or for 
direct tracheal or intravascular injection by diluting the vector to 
approximately 10.sup.6 -10.sup.12 pfu per ml in normal saline and 
delivering (0.5-5 ml) of this solution by the chosen route; whether 
intravenous, intratcheal, or aerosol. If plasmid vectors are utilized, 
approximately 1-2 mg of plasmid DNA is mixed with cationic lipids; for 
example, DOTMA Lipofectin or Lipofectamine in approximate ratios of 1:10 
to 1:100 and delivered intratracheally by bronchoscope or vascularly, 
intravenously or by aerosol administration. 
The efficacy and lung cell specificity of the lung specific vector can be 
assessed in vitro and in vivo. In vitro, H441-4 cells (human bronchiolar 
adenocarcinoma cells that express endogenous human SP-A and SP-B) are 
transfected with viral or plasmid constructs driven by the SP-B promoter 
element (or chimeric element containing TTF-1 and/or HNF-3 .alpha. and 
.beta. binding sites). Approximately 24-48 hours after transfection, 
expression of the chimeric gene is assessed by RNA analysis (S1, RT-PCR, 
or Northern blots), by the synthesis and secretion of the gene products 
which are assessed by ELISA, Western blot, immunocytochemistry or by 
biological assays, or by immunoprecipitation of .sup.35 S 
cysteine/methionine labeled proteins assessed by autoradiography after 
SDS-PAGE of either media or cell lysates obtained from the transfected 
cells. In one embodiment, H441-4 cells and control HeLa cells (which 
normally do not express human surfactant protein B) are transfected with 
the viral or plasmid constructs hereinabove described, and evaluated for 
expression as described in Bohinski, et al., J. Biol. Chem., Vol. 268, 
pgs. 11160-11166 (1993). Cell specificity of the chimeric SP-B promoter 
driven transgene is assessed by transfection of non-lung cells, such as 
3T3 fibroblasts, HeLa, CHO, or other appropriate mammalian cell systems. 
To test the efficacy of and specificity of the SP-B driven constructs, the 
recombinant virus is instilled intratracheally, via tracheal cannulae or 
by aerosolization or by direct injection in 50 .mu.l of diluent containing 
1.times.10.sup.8 -1.times.10.sup.11 pfu per ml of the adenovirus, 
administered into the trachea of rodent or other mammalian models, such as 
mice, Cotton rats or hamsters. Larger volumes are utilized for larger 
animals, depending on the expected sites of delivery. After 24-72 hours, 
lungs are excised, the transfer of the gene assessed by measuring the 
recombinant protein in lavage, or lung homogenates, by ELISA, Western 
blot, or by biological assay. organ specificity can be assessed readily by 
RNA analysis (S1 nuclease, RT-PCR, Northern blot or by in situ 
hybridization). Alternatively, immunocytochemistry, comparing lung and 
other tissues is utilized to assess the specificity and abundance of 
expression of the chimeric gene. Constructs expressing in a lung 
epithelial cell-specific or selective manner and providing appropriate 
abundance of gene transcripts, which are likely to result in genetic 
correction of the metabolic defect targeted by the vector, are utilized 
for clinical testing and use. 
In one embodiment, Av1SPB3 is administered in vivo to the lungs of mice, 
followed by in situ hybridization of sense (control) and antisense (SPB 
specific) cRNA probes to lung tissue as described in Yei, et al., Am. J. 
Cell. and Molec. Biol., (in press). 
Example 4 
Identification of TTF-1 Binding Sites in Murine Surfactant Protein A (SP-A) 
Gene 
Plasmid Constructions and Site-directed Mutagenesis-5' Flanking sequences 
of the mouse SP-A gene (base pairs -255 to +45) were isolated from 
pCPA-1.4 (Korfhagen, et al., Am. J. Physiol., Vol. 263, pgs. L546-L554 
(1992)) using polymerase chain reactions and linker primers to create a 
5'-HindIII and 3'-PstI sites. The product was digested with HindIII and 
PstI and cloned into pCPA-0 to generate pCPA-0.3. To generate the TTF-1 
site mutants, the pCPA-0.3 was used as template for the polymerase chain 
reactions. Oligomers were made to each of the three TTF-1 binding sites, 
replacing each with a restriction enzyme sequence. The TTF-1 site located 
at position -223 to -218 was changed to a SalI site, the site located at 
-200 to -195 was changed to a NcoI site, and the TTF-1 site at position 
-190 to -185 was changed to a BamHI restriction site. These oligomers were 
then used in polymerase chain reactions with pCPA-0.3 as template and 
linker primers used to generate the wild-type sequences. The products were 
then digested with appropriate endonucleases and cloned into pCPA-0. These 
SP-A promoter-chloramphenicol acetyltransferase (CAT) fusion plasmids were 
designated pCPA-0.3T-1,3, pCPA-0.3T-3, and pCPA-0.3T-3,4 and their 
identities were confirmed by dideoxy sequencing of M13 mp19 templates. The 
sequence originally published for the 5'-flanking sequence was incorrect 
at position -4. There is no C in that position. Therefore, all sequences 
in this example differ by -1 from the published sequences. (Korfhagen, et 
al., 1992). 
Cell Culture, Transfection, and Reporter Gene Assays--Cells were cultured 
and transfection experiments were performed essentially as previously 
described (Bohinski, et al., Mol. Cell. Biol., Vol. 14, pgs. 5671-5681 
(1994)). MLE-15 cells were derived from lung tumors produced in transgenic 
mice expressing SV40 large T antigen (SV40 TAg) driven by the 
lung-specific human SP-C promoter (Wikenheiser, et al., Proc. Nat. Acad. 
Sci., Vol. 90, pgs. 11029-11033 (1993)). MLE-15 is a clonal cell line 
expressing SP-A, SP-B, and SP-C. For TTF-1 transactivation experiments 
with HeLa cells, 10-cm dishes were treated with precipitates prepared by 
using 7.5 pmol of promoter-CAT fusion plasmid, 4 pmol of pCMV-.beta.gal, 
and 1 pmol of either the empty expression vector (pRc/CMV) (Invitrogen), 
which includes a CMV promoter, a multiple cloning site and a neomycin 
resistance gene, or an expression vector containing the entire TTF-1 open 
reading frame (pCMV/TTF-1) as previously described (Bohinski, et al., 
1994). Cell lysates were assayed for .beta.-galactosidase and CAT 
activities. To minimize variability, cells used for each construct were 
plated at the same density, transfected, and harvested at the same time. 
Nuclear Extract Preparation--MLE-15 nuclear extracts were prepared by using 
a modified extract procedure as described by Bohinski et al., 1994. 
Nuclear extraction was performed at +4.degree. C. or on ice with ice-cold 
reagents. Confluent monolayers from six 10-cm-diameter dishes were washed 
twice with 10 ml of ice-cold phosphate-buffered saline (pH 7.2) and 
harvested by scraping into 1 ml of phosphate-buffered saline. Cells were 
pelleted in a chilled 1.5-ml microcentrifuge tube at 3000 rpm for 5 min. 
The pellet was washed once in phosphate-buffered saline and repelleted as 
described above. The cell pellet was resuspended in 1 cell volume of fresh 
(lysis) buffer A (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 1.5 MM 
MgCl.sub.2, 0.2% (v/v) Nonidet P-40, 1 mM dithiothreitol, 0.5 mM 
phenylmethylsulfonyl fluoride). Cells were lysed in this buffer during a 
5-min incubation with occasional vortexing. The nuclear pellet was 
obtained by centrifugation at 3000 rpm for 5 min and was resuspended in 1 
volume of fresh (extract) buffer B (20 mM Hepes (pH 7.9), 420 mM NaCl, 0.1 
mM EDTA, 1.5 mM MgCl.sub.2, 25% (v/v) glycerol, 1 mM dithiothreitol, 0.5 
mM phenylmethylsulfonyl fluoride). Nuclei were extracted during a 10-min 
incubation with occasional gentle vortexing. Extracted nuclei were 
pelleted by centrifugation at 14,000 rpm for 10 min. The supernatant was 
saved as the extracted nuclear protein. Extracts typically contained 
5.0-10.0 .mu.g of nuclear protein per .mu.l. Nuclear extracts were quick 
frozen and stored at -80.degree. C. 
Synthetic oligonucleotides--Single-stranded oligonucleotides were 
synthesized on an ABI oligonucleotide synthesizer by the Oligonucleotide 
Synthesis Core Facility, Children's Hospital Medical Center. 
Single-stranded oligonucleotides were annealed at 10 .mu.M in 100 .mu.l 
annealing buffer M (10 mM Tris (pH 7.5), 10 mM MgCl.sub.2, 50 mM NaCl) in 
a 95.degree. C. dry heat block and then slowly cooled to room temperature. 
The absorbance of 260 nm (A.sub.260) was determined, and dilutions of this 
mixture were made in TE (10 mM Tris (pH 8.0), 1 mM EDTA). These 
double-stranded oligomers were either used directly as cold competitors in 
an electrophoretic mobility shift assay (EMSA) or gel purified for 
labeling. For use as a probe in the EMSA, 20 .mu.l of the annealed 
oligomer was gel purified using a 4% Biogel and a MERmaid kit as specified 
by the manufacturer (Bio 101, Inc.). The A.sub.260 was determined, and 1.5 
pmol of annealed and gel-purified oligonucleotide was end labeled using 
[.gamma..sup.32 P]ATP and T4 polynucleotide kinase. End-labeled probe was 
purified from unincorporated nucleotide by using a Pharmacia nick column 
and recovered in 400 .mu.l of TE. 
EMSA--Nuclear extracts (5.0-10.0 .mu.g of protein) and unlabeled 
oligonucleotide competitors were preincubated in 12.5 .mu.l of buffer 
containing 12 mM Hepes (pH 7.9), 4 mM Tris-Cl (pH 7.9), 50 mM KCl, 5 mM 
MgCl.sub.2, 1 mM EDTA, 1 mM dithiothreitol, 75 ng/.mu.l poly(dI-dC) 
(Boehringer Mannheim), 0.2 mM phenylmethylsulfonyl fluoride for 10 min on 
ice. Radiolabeled oligonucleotide or DNA fragments were added to the 
mixture and incubated an additional 20 min. on ice. For antibody 
supershift assays, 1 .mu.l of TTF-1 antibody was added following addition 
of the nuclear extract and incubated as above. The TTF-1 antibody was 
previously described by Lazzaro et al., Development, Vol. 113, pgs. 
1093-1104 (1991). Recombinant TTF-1 homeodomain protein (TTF-1 HD) was 
expressed in Escherichia coli and used as described by Damante and Di 
Lauro, Proc. Nat. Acad. Sci., Vol. 88, pgs. 5388-5392 (1991). Assays were 
performed with 1 .mu.l of TTF-1 HD in place of nuclear extract. The 
protein-DNA complexes were resolved from free probe by nondenaturing 
polyacrylamide gel electrophoresis with 5% gels (29:1, 
acrylamide/bisacrylamide; 0.5.times.TBE (44.55 mM Tris, 44.5 mM borate, 1 
mM EDTA, pH 8.3); 2.5% (v/v) glycerol; 1.5 mm thick) were electrophoresed 
in 0.5.times.TBE buffer at constant current (30 mA) for approximately 90 
min. Gels were blotted to Whatman 3MM paper, dried under vacuum, and 
exposed to x-ray film for 1 h at -80.degree. C. with an intensifying 
screen. 
Cell-specific Activity of SP-A Gene Constructs in Murine Lung Epithelial 
Cells (MLE-15 Cells)--SP-A is expressed specifically in the distal 
pulmonary epithelium. To determine sequences controlling SP-A gene 
expression, MLE-15, 3T3, H441, and HeLa cells were transfected with 
plasmids containing murine SP-A flanking sequences and the bacterial 
reporter gene, CAT. (FIG. 23) As shown in FIG. 23, to the left, the 
5'-flanking region and portion of exon 1 of the mouse surfactant protein A 
(SP-A) gene are depicted. Potential binding sites for TTF-1 or hepatocyte 
nuclear factor-5 (HNF-5) are depicted above the line. Nucleotide positions 
are depicted below the line, and cat indicates the position of the 
chloramphenicol transferase gene. To the right of each clone, CAT activity 
is plotted relative to the promoterless plasmid, pCPA-O. The transfection 
data are representative of at least five separate transfections for MLE-15 
and 3T3, and two experiments for HeLa and H441. Presented data were 
calculated from two experiments with triplicate samples for each constrct 
(n=6). Values represent mean.+-.standard error. The values of pCPA 1.4 and 
pCPA 0.3 in HeLa or H441-4 cells were less than for pCPA-O and therefore 
are not distinguished in the graph. 
MLE-15 cells are murine lung epithelial cells expressing SP-A, -B, and -C 
(Wikenheiser, et al., Proc. Nat. Acad. Sci., Vol. 90, pgs. 11029-11033 
(1993)). Plasmids containing SP-A sequences from nucleotides -255 to +45 
from the start of transcription were approximately 20-fold more active 
than the promoterless plasmid pCPA-0. A larger construct containing 
sequences from -1401 to +45 was approximately 2-3-fold more active than 
the -255 to +45 construct in MLE-15 cells. The SP-A-CAT constructs were no 
more active in 3T3, H441, or HeLa cell lines than pCPA-O. 
Murine SP-A Sequences Are Transactivated by TTF-1 in HeLa Cells--The 
nucleotide sequences of the proximal 5'-flanking region of murine SP-A 
gene contained consensus motifs predicting TTF-1 binding. To determine 
whether these sequences were transactivated by TTF-1, deletion constructs 
of the 5'-flanking region of the murine SP-A gene were cotransfected into 
HeLa cells with pCMV-TTF-1 (FIG. 24). As shown in FIG. 24, CAT activity is 
plotted relative to the activity of the promoterless plasmid. Activity was 
assessed with and without cotransfection with pCMV-TTF-1. CAT activity 
from pCPA-0.1 or pCPA-O was not appreciably altered by cotransfection with 
pCMV-TTF-1. The transfection data are representative of four separate 
transfections. Presented data were calculated from two experiments with 
triplicate samples for each construct (n=6). Value represents 
mean.+-.standard error. Absence of an error bar means that the standard 
error was too small to be indicated on the graph. The standard error was 
not greater than +20% on those lanes. 
The SP-A-CAT construct containing -255 to +45 was approximately 15-fold 
more active after transfecting cells with the TTF-1 expression vector than 
with a promoterless plasmid, pCPA-O. Although consensus motifs for TTF-1 
were present in the region from -1401 to -256, this construct was only 
slightly more active (20- versus 15-fold) than the SP-A-CAT construct 
containing sequences from -255 to +45. Sequences from -57 to +45 were not 
transactivated by TTF-1 but retained low level promoter activity in HeLa 
cells. 
TTF-1 Binds to the SP-A Gene--Since sequences from -255 to +45 markedly 
activated CAT expression in transfected MLE-15 cells, we focused our 
studies to this region. To determine whether the TTF-1 binding motifs 
bound TTF-1, EMSAs were performed with recombinant TTF-1 homeodomain 
protein and double-stranded DNA fragments from sequences -231 to -168 as 
depicted in FIG. 25. As shown in FIG. 25, the corresponding nucleotide 
positions of the SP-A 5'-flanking region are listed with the top sequence 
(probe A). The positions of the TTF-1 binding motifs are underlined and 
numbered 1, 2, 3, or 4. 
The TTF-1 homeodomain had been shown to bind to TTF-1 motifs within the 
SP-B gene (Bohinski, et al., 1994). TTF-1 homeodomain protein bound the 
SP-A DNA fragments in mobility shift assays. Four distinct TTF-1-DNA bands 
were identified with probe A (base -231 to base -168), two with probe B, 
and one with probes C and D (FIG. 26). As shown in FIG. 26, letters A-D at 
the top of the figure indicate the probe used in each lane. Probe means 
the presence (+) of the labeled oligomer in each lane. TTF-1 is the 
presence (+) or absence (-) of TTF-1 homeodomain. With probe A, four bands 
were detected; two were detected with probe B, and one each was detected 
with probes C and D. The slowest migrating band for probe A is faint in 
this exposure, so its position is marked with an arrow. Free probe is 
marked with an arrowhead. 
The heterogeneity of complex formation with this region of the SP-A gene 
supported the concept that probes A and B contained multiple TTF-1 binding 
sites. 
MLE-15 Cells Contain TTF-1 Nuclear Proteins Interacting with SP-A 
Sequences--To determine if MLE-15 extracts contained TTF-1 protein that 
bound to SP-A gene sequences, EMSAs were performed with MLE-15 extracts 
and a polyclonal antibody to TTF-1 (FIG. 27). This antibody was raised to 
three peptides of TTF-1 as described by Lazzaro et al., 1991. In previous 
studies of Bohinski et al., 1994, this antibody caused a supershift in 
EMSAs with the SP-B gene. As shown in FIG. 27, Letters B-E at the top of 
the figure indicate the probe used in each lane. Probe means the presence 
(+) of labeled oligomer in each lane. MLE-15 means the presence (+) of 
nuclear extracts; .alpha.-TTF-1 means the presence (+) or absence (-) of 
TTF-1 antibody. Position of major bands are marked with arrowheads, and 
the supershifted band is marked with an arrow. Exposures are 1 hr. at 
-80.degree. C. for B, 18 hrs. at room temperature for C, 30 min at 
-80.degree. C. for D, and 24 hrs. at room temperature for E. 
As assessed by EMSA (FIG. 27), TTF-1 in nuclear extracts of MLE-15 cells 
bound to SP-A sequences. Since fragment B formed two bands with TTF-1 
(FIG. 26), probe E was used to identify a second TTF-1 binding site. 
Nuclear extracts from MLE-15 cells bound to the E gene fragment, 
consistent with the presence of a distinct TTF-1 binding site in this 
region. Thus, four distinct TTF-1 binding sites were identified in the 
SP-A gene fragment -231 to -168. 
Mutation of TTF-1 Consensus Motifs Decreases Activity in MLE-15 
Cells--Interpretation of DNA footprint analysis of -231 to -168 was 
complicated by the multiple protein-DNA interactions in the region that 
obscured precise identification of footprint sites (data not shown). 
Therefore, the function of some of the TTF-1 binding sites in the SP-A 
gene was determined in SP-A-CAT constructs, in which multiple base changes 
were introduced into the likely TTF-1 sites. Mutations in each of three 
TTF-1 binding sites reduced expression of the SP-A-CAT constructs in 
transfected MLE-15 cells about 10-fold and reduced transactivation in HeLa 
cells (FIG. 28). 
As shown in FIG. 28, Panel A is a schematic representation of the TTF-1 
sites with mutated sequences indicated with asterisks. Panel B is 
transfection analysis of MLE-15 cells, and relative CAT activity is 
presented relative to the activity of the promoterless pCPA-O plasmid. The 
transfection data are representative of four separate transfections. 
Presented data were calculated from two experiments with triplicate 
samples for each construct (n=6). Value represents mean.+-.standard error. 
Panel C is an autoradiogram of representative CAT assays of MLE-15 cells. 
Each construct is presented in duplicate. Panel D is transactivation with 
TTF-1 in HeLa cells. The transfection data are representative of two 
separate transfections. Relative CAT activity is presented relative to the 
activity of the promoterless pCPA-O plasmid. Presented data were 
calculated from both experiments with triplicate samples for each 
construct (n=6). Value represents mean.+-.standard error. Panel E is an 
autoradiogram of representative CAT assays of HeLa cells. Each construct 
is presented in duplicate. Absence of error bars means that the standard 
error was too low to be represented in the graph. Standard error did not 
exceed .+-.20% in those lanes. 
TTF-1 site 3 appeared to have the highest affinity for TTF-1 in EMSA (note 
FIG. 27), so it was tested separately. Mutation of sites 1 or 4 in 
combination with site 3 did not markedly reduce the effect of the site 3 
mutation. Site 2 had the least affinity for TTF-1 and was therefore not 
tested by mutational analysis. The combination of EMSA and mutational 
analysis supports the model that each of the sites indicated in FIGS. 25 
and 28 is required for full transcriptional activity of SP-A sequences in 
MLE-15 cells. 
Example 5 
Identification of TTF-1 Binding Sites in Distal Promoter Region of Human 
Surfactant 
Protein B (SP-B) Gene 
Plasmid Constructions and PCR-mediated Site-Directed Mutagenesis 
The human SP-B promoters with various length and regions were generated by 
polymerase chain reaction (PCR) using Taq DNA polymerase (BRL), synthetic 
oligonucleotide primers and the p.DELTA.5'-650 SP-B CAT construct as a 
template (Bohinski, et al., J. Biol. Chem., Vol. 268, pgs. 11160-11166 
(1993)). The upstream primer with the Mlu I site for the B-281 construct 
is 5'-CGCACGCGTGAACATGGGAGTCTGGGCAGG. (SEQ ID NO.: 40) The upstream primer 
with the Mlu I site for the B-500 construct is 
5'-CGCACGCGTCAGAAGATTTTTCCAGGGGAA. (SEQ ID NO.: 41) The downstream primer 
with the Xho I site for the B-281 and the B-500 construct is 
5'-GCGCTCGAGCCACTGCAGCAGGTGTGACTC. (SEQ ID NO.: 42) The upstream primer 
with the Mlu I site for the SV40-P F construct is 
5'-CGCACGCGTCAGGGCTTGCCCTGGGTTAAG. (SEQ ID NO.: 43) The downstream primer 
with the Xho I site for the SV40-P F construct is 
5'-GCGCTCGAGGCCTGGGTGTTCCCCTCCCAT. (SEQ ID NO.: 44) The upstream primer 
with the Mlu I site for the SV40-P R construct is 
5'-CGCACGCGTGCCTGGGTGTTCCCCTCCCAT. (SEQ ID NO.: 45) The downstream primer 
with the Xho I site for the SV40-P R construct is 
5'-GCGCTCGAGCAGGGCTTGCCCTGGGTTAAG. (SEQ ID NO.: 46) The PCR products were 
digested with Mlu I and Xho I restriction enzymes (BRL) and ligated with 
Mlu I/Xho I digested pGL2-B or pGL2-P luciferase reporter plasmids 
(Promega). The oligonucleotide sequences for the PCR II-C construct are: 
upstream primer 5'-CAGGGCTTGCCCTGGGTTAAG; (SEQ ID NO.: 47) downstream 
primer 5'-GCCTGGGTGTTCCCCTCCCAT. (SEQ ID NO.: 48) The PCR product was 
directly subcloned into the PCR II vector as described by the manufacturer 
(Invitrogen). 
To generate the site-specific mutants of B-500 construct at the TTF-1 
binding sites, two steps of PCR were conducted. For the first PCR, proper 
mutant PCR oligonucleotides were synthesized with mutations at the 
position indicated in FIG. 34A. The mutant primers were mixed with the 
pGL2-B vector primer GLprimer 1 and GLprimer 2 (Invitrogen) to make two 
sets of PCR products that were subsequently purified by low melting point 
(LMP) agarose gel electrophoresis and the QIAquick gel extraction kit. The 
purified PCR products were then mixed together along with GLprimer 1 and 
GLprimer 2 primers for the second PCR. The second PCR products were 
digested with Mlu I/Xho I restriction enzymes for 3 hrs. at 37.degree. C. 
The DNA fragments (553 bp) with Mlu I and Xho I flanking sites at each end 
were purified by LMP gel electrophoresis as described above and ligated 
into the Mlu I/Xho I digested pGL2-B plasmid to generate B-500 Ba.sup.m, 
B-500 Bb.sup.m and B-500 Bcm mutant luciferase constructs. The correctness 
of all the wild type and mutant plasmid constructs were confirmed by DNA 
sequencing. 
Cell culture, transfection and reporter gene assays 
H441 cells were maintained in RPMI medium (BRL) supplemented with 2 mM 
glutamine and 10% fetal calf serum (BRL). One day before transfection, 
5.times.10.sup.5 cells were seeded into 60 mm dishes. Each dish was 
transfected with 12.5 .mu.g of total plasmid DNA using the calcium 
phosphate precipitation method and incubated in Dulbecco's Modified Eagle 
medium overnight. The next day, media was changed to RPMI and the cells 
incubated for 2 days prior to assay. Cell lysis and luciferase assays were 
performed using the luciferase assay system purchased from Promega. The 
light units were assayed by luminometry (monolight 2010, Analytical 
Luminescence Laboratory, San Diego, Calif.). Transfection efficiency was 
normalized to .beta.-galactosidase activity. Multiple transfections (n=2 
to 8) were carried out for each experiment and the mean values were used 
for data presentation. Standard deviations were generally less than 20%. 
Plasmids pCMV-Rc and pCMV-TTF-1 were kind gifts from Dr. R. Di Lauro, 
Stazione Biologic, Naples, Italy. 
Nuclear extracts and EMSA 
H441 cells were grown in 75 mm flasks. Before harvesting, cells were washed 
twice in Hank's solution (HBSS). The cell pellet was then resuspended in 5 
volumes of lysis buffer (50 mM Tris-Cl, 100 mM NaCl, 5 mM MgCl.sub.2 and 
0.5% (vol/vol) Nonidet P-40) for 5 minutes on ice. After centrifugation, 
the supernatant was saved as cytoplasmic protein extract. The nuclear 
pellet was resuspended in 100 .mu.l of nuclear buffer (0.5 M KCl, 20 mM 
Tris-Cl, pH 7.6, 0.2 mM EDTA, 1.5 mM MgCl.sub.2, 25% glycerol and 1 mM 
DTT) and incubated on ice for 30 min. The resulting DNA pellet was spun 
down and the supernatant was used as nuclear extract (NE). Protein extract 
(5 .mu.g) was used for electrophoresis mobility shift assay (EMSA) as 
described previously (Yan, et al., J. Biol. Chem., Vol. 265, pgs. 
20188-20194 (1989)). Recombinant rat TTF-1 homeodomain (HD) was the kind 
gift from Dr. Di Lauro. The probes for EMSA were made from either the 
synthetic oligonucleotides or the PCR product (hSP-B -439/-331 fragment). 
Expression of SP-B, SV40 and TK promoters in H441 cells. 
As shown in FIG. 29A, the underlined nucleotide consensus sequences (CAAG) 
are the putative TTF-1 binding sites. Bars Ba, Bb, and Bc represent the 
regions used to design the oligonucleotides for the mutagensis study 
described hereinbelow. 
FIG. 29B depicts schematics of the plasmid constructs used in this example. 
B is a promoterless pGL2-B luciferase reporter vector. B-218 is a pGL2-B 
vector containing the human SP-B promoter region from -218 to +41 bp. 
B-500 is a pGL2-B vector containing the human SP-B promoter region from 
-500 to +41 bp. SV40-P is a pGL2-B vector containing the SV40 promoter. 
SV40-P F is the SV40 vector fused with the human SP-B distal promoter 
region from -439 bp to -331 bp, with the enhancer in the forward 
orientation. In SV40-P R, the enhancer is in the reverse orientation. 
PCRII-C is the PCRII vector containing the human surfactant protein B 
distal promoter region from -439 bp to -331 bp and the proximal promoter 
from bp -218 to bp +41 at the EcoRI site. 
FIG. 30 shows SP-B promoter activity in H441 cells. Plasmid DNA (12.5 
.mu.g/60 mm dish) was used to transfect H441 cells. Cells were transfected 
with 5 .mu.g pCMV-.beta. gal (a plasmid including a B-galactosidase gene 
under the control of a CMV promoter) and 7.5 .mu.g of B (lane 1), SV40-P 
(lane 2), TK (lane 3), B-218 (lane 4), and B-500 (lane 5). The TK vector 
contains a luciferase gene under the control of a Herpes Simplex Virus 
thymidine kinase (TK) promoter. Such vector was constructed by digesting 
pBLCAT5 (Boschart, et al., Gene, Vol. 110, pgs. 129-130 (1992) with BamHI 
and BglII in order to obtain a 165 bp fragment including the Herpes 
Simplex Virus thymidine kinase promoter. This fragment then was cloned 
into BamHI and BglII digested pGL2-B (also sometimes known as pGL2-Basic) 
to form the TK plasmid vector construct. The luciferase assays were 
carried out in duplicate two days after transfection. 
When the constructs including the B-218 and B-500 promoters were compared 
with the SV40 and TK promoters in H441 cells using transient transfection 
assays, both B-218 and B-500 constructs were more active than the SV40 and 
TK promoters (FIG. 30). Activity of B-500 was 3-4 fold greater than B-218 
indicating a potential enhancer element located in the distal upstream 
region. 
Transcriptional activity and DNA Protein binding of hSP-B (-439 to -331) 
Nucleotide sequences in the 5'-flanking regions of the human and mouse SP-B 
genes share 95% identity from -439 to -331 bp (human) and -382 to -282 bp 
(mouse). Deletion of this region in the mouse SP-B gene dramatically 
reduced the transcriptional activity (50 fold reduction) as assayed by 
transient transfection of the mouse lung epithelial (MLE-15) cell line, 
using the chloramphenicol acetyl transferase (CAT) reporter gene 
(Whitsett, et al, unpublished observations). In order to determine the 
biological function of the stimulatory element in the human gene, the 
hSP-B(-331/-439) sequence was subcloned into the PCR II vector. The final 
construct PCR II-C (FIG. 29B g) was generated using the standard PCR 
procedure. Transient transfection of the B-500 construct with an excess 
amount of PCR II-C competitor plasmid reduced transcriptional activity 
from B-500 to the level of B-218 activity (FIG. 31A, lane 4), compared to 
the 4 fold activity without the PCR II-C competitor. 
In this experiment, the results of which are shown in FIG. 31A, total 
plasmid DNA of 12.5 .mu.g/60 mm dish was used in transfection, which 
contains 2.5 .mu.g pCMV-.beta.gal, 1.5 .mu.g of B (lane 1), B-218 (lane 
2), B-500 (lane 3 and 4) and 8.5 .mu.g of PCR II-C (lane 4) or PCR II 
vector (lane 1, 2 and 3). This figure represents two separate experiments, 
each assay performed in duplicate. Mean values (fold stimulation) and 
standard deviations are: lane 1, 0.+-.0; lane 2, 1.+-.0, lane 3, 
3.7.+-.0.8; lane 4, 1.2.+-.0.17. 
The competition experiments suggested the presence of trans-acting factors 
that interact with the hSP-B(-331 to -439) element. EMSA was used to 
examine the nuclear proteins binding to the hSP-B -331 to -439 region. In 
such experiment, the results of which are shown in FIG. 31B, the 
hSP-B(-439/-331) enhancer fragment was end-labeled by [.gamma.-.sup.32 P] 
ATP with T4 kinase. The probe with 20,000 dpm was incubated with 5 .mu.g 
of H441 cytoplasmic (C) or nuclear (N) extracts and run on a 4% 
polyacrylamide gel. Only one DNA-binding protein (BP) complex was observed 
in the nucleus after gel electrophoresis and autoradiography. 
No shift in mobility was observed with the cytoplasmic fraction from H441 
cells (FIG. 31B). 
TTF-1 binds to the hSP-B(-439/-331) fragment of the human SP-B gene 
Three distinct CAAG motifs (Damante, et al., Nucleic Acids Research, Vol. 
22, pgs. 3075-3083 (1994)) were present in the hSP-B(-439/-331) fragment. 
This fragment was tested to determine whether this fragment contains TTF-1 
binding site(s) (FIG. 29A). DNA oligonucleotide F.sub.1, a TTF-1 binding 
site previously identified in the proximal element of the human SP-B gene 
(Bohinski, et al., Mol. Cell. Biol., Vol. 14, pgs. 5671-5681 (1994)), was 
used as a competitor in EMSA to test whether the nuclear protein binding 
to the hSP-B(-439/-331) fragment was TTF-1. In one EMSA experiment, the 
results of which are shown in FIG. 32A, radio-labeled human SP-B (-439 to 
-331 bp) enhancer probe (35,000 dpm) was incubated with 5 .mu.g of H441 
cytoplasmic (C) or nuclear (N) extracts in the presence of no competitor 
(-), self-competitor (S), or F, fragment (F.sub.1 contains other TTF-1 
binding sites of the human SP-B gene) and run on a 4% polyacrylamide gel. 
The DNA-binding protein (BP) complex was inhibited by S or F.sub.1 DNA 
competitors. 
FIG. 32A demonstrates that the specific interaction between the H441 
nuclear protein and the radio-labeled hSP-B(-439/-331) fragment was 
inhibited by adding 50 fold molar excess of F.sub.1 fragment or self 
competitor. This protein-DNA complex was retarded with TTF-1 antibody in 
the supershift analysis (data not shown). 
In another EMSA experiment, the results of which are shown in FIG. 32B, 
radio-labeled hSP-B(-439/-331) enhancer probe (40,000 dpm) was incubated 
with 3 .mu.g of purified recombinant TTF-1 homeodomain protein in the 
presence of no competitor (-), self-competitor (S), F.sub.1 fragment 
(F.sub.1) and the F.sub.2 fragment (F.sub.2 contains an HNF-3 binding 
site) of the human SP-B gene and separated on 4% polyacrylamide. Three 
protein-DNA complexes (a, b and c) were detected by the EMSA. 
When the radio-labeled hSP-B(-439/-331) fragment was incubated with the 
purified TTF-1 HD protein, three protein-DNA complexes were observed (FIG. 
32B), lane 1), consistent with the presence of three TTF-1 binding sites 
in the DNA fragment -439/-331. These TTF-1 complexes were inhibited by 
adding 50 fold molar excess of self-competitor and the F.sub.1 fragment 
(FIG. 32B, lane 2 and 3), confirming that TTF-1 interacts with multiple 
binding sites in the hSP-B(-439/-331) fragment. 
hSP-B(-439/-331) activates transcription from SV40 and SP-B promoters 
pCMV-TTF-1 was co-transfected with B-218 and B-500 into H441 cells. 
pCMV-TTF-1 activated transcription of B-218 approximately 4 fold. In one 
experiment, the results of which are shown in FIG. 33A, H441 cells were 
transfected with plasmid DNA (12.5 .mu.g/60 mm dish) containing 2.5 .mu.g 
pCMV-.beta.gal, 5.mu.g of B (lane 1, 2), B-218 (lane 3, 4), B-500 (lane 5, 
6) and 5 .mu.g of pCMV-Rc (lane 1, 3, 5) or pCMV-TTF-1 (lane 2, 4, 6). 
B-218 activity is set as 1. TTF-1 transactivated both B-218 and B-500. The 
figure represents four separate experiments, each assay performed in 
duplicate. Mean values of fold stimulation and standard deviations are: 
lane 1, 0.+-.0; lane 2, 0.016.+-.0; lane 3, 1.+-.0; lane 4, 4.2.+-.0.57; 
lane 5, 3.6.+-.0.47; lane 6, 12.3.+-.1.4. 
pCMV-TTF-1 further activated B-500 transcription (11 fold), FIG. 33A. Since 
there are two active TTF-1 sites in B-218, it was not possible to discern 
the distinct contributions of the activity from the three putatitive TTF-1 
sites in the hSP-B(-439/-331) fragment from those in the proximal 
(F.sub.1) element located -111 to -73 bp. The hSP-B(-439/-331) fragment 
was therefore isolated and ligated to an SV40 promoter-luciferase 
construct in the forward and reverse orientation producing SV40-P F and 
SV40-P R, FIG. 29B. 
Another experiment (results are shown in FIG. 33B) thus was conducted 
similar to that hereinabove described, wherein the results were shown in 
FIG. 33A, except that construct B (lane 1, 5), SV40-P (lane 2, 6), SV40-P 
F (lane 3, 7) and SV40-P R (lane 4, 8) were co-transfected with pCMV-Rc 
(lane 1, 2, 3, 4) or pCMV-TTF-1 (lane 5, 6, 7, 8). SV40 activity is set as 
1. TTF-1 transactivated both SV40-P F and SV40-P R. The figure represents 
two separate experiments, each performed in duplicate. Mean values and 
standard deviations are: lane 1, 0.+-.0; lane 2, 1.+-.0; lane 3, 
3.5.+-.0.24; lane 4, 8.9.+-.0.24; lane 5, 0.+-.0; lane 6, 1.9.+-.0.3; lane 
7, 8.3.+-.0.38; lane 8, 18.1.+-.1.9. The hSP-B(-439/-331) fragment 
stimulated the SV40 promoter transcriptional activity in both 
orientations. SV40-P R was more active than SV40-P F, FIG. 33B. 
Co-transfection of H441 cells with pCMV-TTF-1 increased SV40-P F activity 
9 fold and Sv40-P R activity 19 fold, FIG. 33B. 
Mutations in the hSP-B(-331/-439) abolished or reduced the TTF-1 response 
To confirm further that the putative TTF-1 binding to the sites in the 
hSP-B(-439/-331) fragment mediated transactivation, three wild type TTF-1 
sites and three mutant oligonucleotides were synthesized (FIG. 34A), 
radio-labeled and incubated with recombinant TTF-1 homeodomain (HD) 
protein and separated by EMSA. As shown in FIG. 34A, the core nucleotides 
(CAAG) of the TTF-1 binding sites were changed to ATTC in the mutants as 
underlined. The locations of the Ba, Bb, and Bc oligonucleotides in the 
hSP-B(-439/-331) enhancer fragment are indicated in FIG. 29A. 
In the EMSA experiment, the results of which are shown in FIG. 34B, 
oligonucleotides were end-labeled with T4 kinase. 
Probes (100,000 dpm) were incubated with 2 .mu.g of TTF-1 purified 
recombinant homeodomain and separated on 4% polyacrylamide gel and 
subjected to autoradiography. w is for wild type oligonucleotides and m is 
for mutant oligonucleotides. 
While all three wild type oligonucleotides were shifted by TTF-1 HD, the 
mobility of mutant oligonucleotides was not altered, FIG. 34B. The mutants 
lacking binding to TTF-1 HD were introduced into the B-500 luciferase 
construct. Wild type and mutant B-500 constructs mutated at the positions 
Ba.sup.m, Bb.sup.m, and Bc.sup.m were transfected into H441 cells. 
In this transfection analysis, the results of which are shown in FIG. 34C, 
the wild type B-218 (2 and 8), B-500 (lane 3 and 9) and mutant B-500 at 
Ba.sup.m (lane 4 and 10), Bb.sup.m (lane 5 and 11) and Bc.sup.m (lane 6 
and 12) were transfected into H441 cells and activity assessed by 
luciferase assays. Lane 1 and 7 contained a promoterless construct B. 
Mutations in the TTF-1 binding sites decreased transcriptional activity of 
all three B-500 mutants. This figure represents three separate 
experiments, each performed in duplicate transfections. Mean values of 
fold stimulation and standard deviations are: lane 1, 0.+-.0; lane 2, 
1.+-.0; lane 3, 4.35.+-.0.46; lane 4, 0.9.+-.0.07; lane 5, 1.03.+-.0.18; 
lane 6, 1.9.+-.0.11; lane 7, 0.02.+-.0; lane 8, 3.24.+-.0.48; lane 9, 
10.7.+-.0.93; lane 10, 2.22.+-.0.24; lane 11, 2.89.+-.0.40; lane 12, 
6.12.+-.1.3. 
As illustrated in FIG. 34C, site specific mutations in the B-500 constructs 
decreased transcriptional activity. Mutations at the position Bar and Bbm 
reduced transcription to the level of the minimal promoter (B-218) and 
completely abolished the stimulatory response produced by cotransfection 
with pCMV-TTF-1. Mutation at the position Bc.sup.m only moderately 
impaired activity. Transcription from the hSP-B(-439/-331) fragment was 
therefore highly dependent on TTF-1 binding to the region. 
In the above example, an upstream enhancer sequence was identified in the 
5' flanking region of hSP-B(-439/-331). This distal element is active in 
the context of the proximal SP-B promoter-enhancer region, and also 
stimulates transcription from a minimal SV40 promoter construct regardless 
of the orientation. TTF-1 binds to and activates the enhancer at three 
distinct sites located within the region -439 to -331 of the human SP-B 
gene. This conclusion is based on several observations: 1) TTF-1 HD binds 
to the enhancer sequence and forms three distinct complexes; 2) nuclear 
proteins bind to the upstream SP-B enhancer sequence, and were competed 
off by a known TTF-1 binding sequence (F.sub.1) and supershifted by the 
TTF-1 antibody; 3) pCMV-TTF-1 expression vector stimulated the SP-B and 
the SV40 promoters linked to the upstream SP-B enhancer sequence; and 4) 
mutations at the three putative TTF-1 binding sites on the 
hSP-B(-439/-331) fragment reduced or abolished TTF-1 HD binding 
transcriptional activity. 
Example 6 
Sixty-six cases of lung carcinomas and 48 breast adenocarcinomas from equal 
number of patients were obtained. The lung neoplasms included 54 non-small 
cell carcinomas; 43 adenocarcinomas, 10 squamous cell carcinomas, and one 
adenosquamous carcinoma obtained from either wedge excision, lobectomy or 
pneumonectomy, and 12 small cell carcinomas, all obtained by 
transbronchial biopsy. The breast adenocarcinomas, obtained from 
excisional biopsies, included 41 invasive ductal carcinomas, 4 invasive 
lobular carcinomas, 2 lobular carcinomas in situ and 1 medullary 
carcinoma. The tissues were fixed in 10% neutral formalin and subsequently 
paraffin embedded. Hematoxylin and eosin sections were independently 
reviewed, the diagnoses confirmed, and the histologic differentiation of 
the tumors was obtained according to the World Health Organization 
classification (Am. J. Clin. Pathol., Vol. 77, pg. 123 (1982)). 
Perioperative clinical work-up on the 114 patients studied did not reveal 
information that might have indicated the possibility of additional 
non-pulmonary or breast primary tumors. Blocks containing the predominant 
pattern in each individual case were chosen for immunohistochemical 
studies after review of the hematoxylin and eosin stained slides in order 
to ensure adequate representation of the tumor cells and normal parenchyma 
within each slide. 
Primary antibodies 
Surfactant protein A was detected with rabbit antihuman SP-A antibody 
prepared against the deglycosylated forms of SP-A as previously described 
(McMahon, et al., Obstet. Gynecol., Vol. 70, pg. 94 (1987); Whitsett, et 
al., Pediatr. Res., Vol. 19, pg. 501 (1985)). This SP-A antiserum 
selectively stains normal adult lung tissues, serous cells in 
tracheal-bronchial glands, subsets of nonciliated epithelial cells in the 
conducting airway, and alveolar Type II epithelial cells (Phelps, et al., 
Experimental Lung Res., Vol. 17, pg. 985 (1991); Snyder, et al., Pulmonary 
Surfactant: Biochemical, Functional, and Clinical Concepts, Bourbon, ed., 
pg. 105, Boca Raton, CRC Press (1991)). Staining for surfactant protein B 
utilized antiserum generated against the purified SP-B protein obtained 
from bovine pulmonary surfactant (Stahlman, et al., J. Histochem. 
Cytochem., Vol. 40, pg. 1471 (1992)). This antibody selectively stained 
bronchiolar and alveolar epithelial cells in the distribution pattern 
similar to that of SP-A. Immunostaining of both antibodies was completely 
ablated by pre-incubation of the antisera with purified SP-A or with SP-B, 
respectively. (Stahlman, et al., 1992; McMahan, et al., 1987). Rabbit 
polyclonal antibody against rat TTF-1 was kindly provided by Dr. Roberto 
DiLauro. This antibody was generated against recombinant rat TTF-1 peptide 
(F2) as previously described by Lazzaro et al., Development, Vol. 113, pg. 
1093 (1991). In normal tissue, TTF-1 antibody stained thyroid and 
pulmonary epithelial cells in a highly selective manner in both human and 
murine tissues. 
Immunohistochemistry: 
For immunohistochemical analysis, four micron thick sections were 
deparaffinized in xylene and rehydrated through decreasing concentrations 
of ethanol to water. Microwave heating of the tissue sections to be 
incubated with TTF-1 antibody was performed prior to staining (Pavelic, et 
al., J. Exp. Pathol., Vol. 5, pg. 143 (1990)). This method for antigen 
retrieval was not needed for the tissue sections to be incubated with SP-A 
or SP-B antibodies. No enzymatic pre-treatment was used for any of the 
three antibodies. Sections were immunostained using an indirect 
biotin-avidin method (Hsu, et al., J. Histochem. Cytochem, Vol. 29, pg. 
577 (1981)) on a Ventana 320 automatic immunostainer (Ventana Medical 
Systems, Inc., Tucson, Ariz., USA). The Ventana 320 is a fully 
computerized bar code-driven, self-contained automatic immunostaining 
device that automatically dispenses reagents and controls washing, mixing, 
and heating to optimize immunohistochemical reaction kinetics. Dilutions 
of the antisera for SP-A was 1/500, SP-B was 1/250, and TTF-1 was 1/500. 
Sections of a moderately to poorly differentiated adenocarcinoma of the 
lung known to express SP-A and SP-B and a papillary carcinoma of the 
thyroid stained with TTF-1 antibody served as positive controls. Negative 
controls were prepared by substituting the primary antibodies with 
nonimmune rabbit ascites fluid in parallel sections of study cases. 
Counterstain for TTF-1 was nuclear fast red and for SP-A and SP-B was 
Harris Hematoxylin. 
The results of the immunostains were based on the estimated percentage of 
positive cells as follows: 0, no staining evident; staining of up to 10%; 
staining greater than 10% up to 50%; and staining greater than 50%. The 
results for each of the antibodies are shown in Table I below. The 
intensity of the stains was also independently evaluated: 0, no stain; 1, 
weak; 2, moderate; 3, strong reaction. A particular tumor was considered 
positive if more than 10% of the tumor cells reacted with any intensity. 
Comparison between groups was done using nonparametric testing including 
Chi square. The Odd's ratio was calculated and the 95% confidence interval 
determined by using the method of Gardner, et al., British Medical 
Journal, Vol. 299, pg. 690 (1989). 
Because of the known heterogeneity of lesions in non-small cell carcinomas 
of the lung, diagnostic criteria were established on the basis of the 
pattern of growth and the level of differentiation. The degree of 
glandular formation, homogeneity of glandular architecture, the presence 
of solid areas, level of mitotic activity and the amount of necrosis was 
utilized to classify adenocarcinomas as described previously (Macay, et 
al., Tumors of the Lung, pg. 100, Philadelphia, W. B. Saunders Co. 
(1991)). On the basis of these criteria, 20 well-differentiated (including 
acinar and papillary types), 12 moderately differentiated (acinar and 
papillary types) and 11 poorly differentiated (solid type) adenocarcinomas 
were identified in the patient population. Pure bronchioalveolar 
carcinomas were not available for study. The extent of keratinization, 
degree of cellular pleomorphism and frequency of mitoses were used to 
discriminate and grade squamous cell carcinomas. Poorly differentiated 
carcinomas were also stained for mucicarmine and digested PAS for their 
assignment to either group, adenocarcinoma or squamous cell carcinomas. 
The only adenosquamous carcinomas diagnosed in this study had both 
components well-differentiated by this criteria. Diagnosis of small cell 
carcinomas was made using previously established histologic criteria using 
hematoxylin and eosin stained sections (Carter, Am. J. Surg. Pathol., Vol. 
7, pg. 787 (1983)). The invasive ductal breast carcinomas (n=41) were 
graded using the Page and Anderson criteria, grade II (31 cases) to grade 
III (9 cases) (Elston, Diagnostic Histopathology, Page, et al., eds., 
Edinburgh, Churchill Livingstone, pg. 300 (1987)). 
Immunohistochemistry Results 
The immunohistochemical staining profile for carcinomas of the lung are 
given in Table I below. 
TABLE I 
__________________________________________________________________________ 
Immunohistochemical staining profile for carcinomas of the lung. 
Number of positive cases based on percentage of stained cells. 
Total number of 
positive cases* 
# of Cases 0% 1-10% 11-50% 51-100% (%) 
__________________________________________________________________________ 
SP-A 
Adenocarcinoma 43 16 4 8 15 23 (53%) 
Squamous cell 10 6 2 2 0 2 (20%) 
Adenosquamous 1 0 0 1 0 1 (10%) 
Small cell 12 11 0 0 1 1 (8%) 
SP-B 
Adenocarcinoma 43 13 4 6 20 26 (60%) 
Squamous cell 10 7 3 0 0 0 (0%) 
Adenosquamous 1 0 0 1 0 1 (10%) 
Small cell 12 10 0 2 0 2 (16%) 
TTF-1 
Adenocarcinoma 43 11 0 2 30 32 (74%) 
Squamous cell 10 10 0 0 0 0 (0%) 
Adenosquamous 1 0 0 1 0 1 (10%) 
Small cell 12 2 0 3 7 10 (83%) 
__________________________________________________________________________ 
*Positive case: &gt;10% of tumor cells are immunoreactive. 
SP-A was detected by immunohistochemistry in malignant cells of the tumors 
in 26 out of 54 non-small cell carcinomas of the lung. SP-A staining the 
tumors included 23 adenocarcinomas, 2 squamous cell carcinomas, and one 
adenosquamous carcinoma. The percentage of positive cells staining for 
SP-A is represented in Table I hereinabove. While SP-A rarely stained 
squamous cell carcinomas, the SP-A staining was detected in two of these 
tumors; one well differentiated and the other poorly differentiated. In 
general, SP-A stained the cytoplasm of malignant cells, primarily in a 
vesicular and granular patterns (FIG. 35A). Reactivity of three of the 23 
adenocarcinomas was detected also in the cell membranes and two tumors had 
nucleoli staining. The adenosquamous carcinoma in this example showed 
reactivity in the cytoplasm of the cells and was limited to the glandular 
component of this tumor. SP-A was detected in the non-neoplastic regions 
of the lung in Type II epithelial cells and in the present example, care 
was taken to distinguish trapped non-neoplastic cells within regions of 
tumor. The pattern of staining for SP-A in Type II epithelial cells was 
that of a foam-like appearance. In only one case staining for SP-A was 
noted in the bronchial epithelium. Plasma cells showed immunoreactivity in 
three cases. 
Surfactant Protein B: 
The pattern of staining for surfactant protein B was similar to that of 
SP-A, staining 27 of the non-small cell carcinomas. Of these tumors, 26 
were adenocarcinoma and one was adenosquamous. SP-B was detected in the 
cytoplasm of tumor cells (FIG. 35B). Squamous cell carcinomas were not 
stained with the antiSP-B antibody. AntiSP-B antibodies stained the single 
adenosquamous carcinoma in the more differentiated glandular components of 
the tumor in the manner similar to that of SP-A staining in this tumor. 
The Type II epithelial cells stained strongly for SP-B with cytoplasmic 
vesicular and foam-like staining pattern. Compared to SP-A, the plasma 
cells did not stain with SP-B antibody, but a higher number of nucleoli, 
bronchi, and bronchioles showed positive staining. 
Thyroid Transcription Factor 1: 
AntiTTF-1 antibody stained 33 of 54 non-small cell carcinomas in this 
study. Of these, 32 (74%) of the lung adenocarcinomas stained for TTF-1. 
The single adenosquamous carcinoma in our study stained for TTF-1. TTF-1 
staining was limited to the nuclei and was characterized by a finely 
granular diffuse pattern in the majority of cells (FIG. 35C). 
Occasionally, the most intense areas were located at the periphery of the 
nucleus. Less intense staining of the nucleoli was also observed. TTF-1 
antibodies accentuated nuclear foldings that were present in the tumor 
cell nuclei. The staining of nuclei in benign Type II epithelial cells was 
also prominent. In general, bronchial, bronchiolar, and tracheal 
epithelia, as well as lamina elastica of arterioles, plasma cells, and 
other cellular elements of the lung, were non-reactive for TTF-1. 
Table I summarizes the staining characteristics of the various non-small 
cell carcinomas of the lung. Staining for surfactant proteins SP-A and 
SP-B was typical in the adenocarcinomas but was rarely observed in 
squamous cell carcinoma. In general, when tumors were positive for 
surfactant proteins, the majority of the malignant cells stained 
positively. This was also observed most clearly for TTF-1, where 50% of 
the cells stained for TTF-1. The extent of cellular staining for SP-A and 
SP-B was somewhat less than for TTF-1. 
Pulmonary Adenocarcinomas 
The majority of adenocarcinomas stained for SP-A (53%), SP-B (60%), and 
TTF-1 (74%). The level of cytodifferentiation was correlated with the 
percentage of tumors that were positive for specific stains as noted in 
Table II below, which shows the immunoreactivity of lung adenocarcinomas 
based on histologic grade. There was no correlation between the level of 
differentiation and the staining for surfactant proteins or TTF-1. 
TABLE II 
______________________________________ 
Immunoreactivity of lung 
Adenocarcinomas based on histologic grade 
SP-A SP-B TTF-1 
______________________________________ 
Well differentiated (n = 20) 
11 (55%) 14 (70%) 15 (75%) 
Moderately differentiated 6 (50%) 7 (50%) 8 (67%) 
(n = 12) 
Poorly differentiated 6 (55%) 5 (45%) 9 (82%) 
(n = 11) 
TOTAL 43 23 26 32 
______________________________________ 
Breast Adenocarcinomas 
None of the breast adenocarcinomas stained for SP-B and TTF-1. The benign 
epithelium of a breast in regions of extensive apocrine metaplasia 
demonstrated reactivity to the antiSP-A antibody in the metaplastic cells 
in areas distinct from the tumor. In this case, the tumor did not stain 
for SP-A. However, the SP-A antibody was clearly reactive with cells of 
the tumors of two cases of invasive ductal cell carcinoma, and in one 
invasive lobular carcinoma. In those cases, SP-A reactivity was limited to 
the cytoplasm, but the pattern of staining was different than that seen in 
carcinomas of the lung, being present in a discrete clumped cytoplasmic 
distribution rather than the granular pattern seen in pulmonary 
adenocarcinoma. As in the lung, plasma cells contained SP-A staining that 
was not detected with either SP-B or TTF-1 antibodies. SP-A, SP-B and 
TTF-1 were highly useful in differentiating lung and breast cancer, as 
shown in Table III below. 
TABLE III 
______________________________________ 
Immunoreactivity of lung and breast adenocarcinomas 
Lung vs. Breast 
Lung Breast Sensitivity 
Specificity 
______________________________________ 
SP-A 23/43* 3/48 53% 94% 
SP-B 26/43 0/48 60% 100% 
TTF-1 32/43 0/48 74% 100% 
______________________________________ 
*Number with &gt;10% positive stain/Total number tested 
Small Cell Carcinomas 
Small cell carcinomas of the lung (n=12) were stained 83% of the time with 
TTF-1, wherein TTF-1 immunostaining was located in finely granular and 
diffuse pattern in the nuclei of the tumor cells (FIGS. 36A, B, C). In 
most of the cases of small cell carcinomas, more than 50% of the tumor 
cells were immunoreactive for TTF-1. In contrast, SP-B and SP-A were 
detected with much less frequency. Only one of the tumors expressed SP-A 
and two SP-B, respectively. The SP-A positive small cell carcinoma was 
also stained by antiSP-B and TTF-1 and one case of small cell carcinoma 
reacted with all three antibodies. TTF-1 staining of small cell carcinoma 
reacted to the nucleus in a pattern similar to that in the non-small cell 
carcinomas. 
Immunohistochemical lung epithelial cell selective markers SP-A, SP-B and 
TTF-1 was utilized to distinguish primary pulmonary from breast 
carcinomas. TTF-1 staining included subsets of non-small cell carcinomas 
expressing SP-A and SP-B but also included small cell carcinomas that 
generally lacked staining for the surfactant proteins. All three of these 
markers were highly useful in distinguishing pulmonary from breast 
carcinoma. SP-B and TTF-1 were never detected in breast carcinoma. These 
studies therefore support the concept that TTF-1 likely regulates 
epithelial cell specific gene expression that includes multiple cell 
types, including progenitor cells that may be shared by small and 
non-small cell carcinoma. 
Thus, the finding that SP-B and TTF-1 and SP-A are commonly co-expressed in 
the lung tumors provides support for the general role of TTF-1 in lung 
epithelial cell gene expression. Surprisingly, small cell carcinoma cells, 
a cell type that rarely synthesizes surfactant proteins, commonly 
expressed TTF-1 (83% of cases). Thus, TTF-1 provides a useful role in 
marking both non-small cell and small cell carcinoma arising from the 
respiratory epithelium. The finding that TTF-1 is commonly expressed in 
small cell carcinoma also supports its potential role in the 
differentiation as well as gene expression in the small cell carcinoma 
cell type. 
The present example confirms previous work that demonstrated the presence 
of SP-A in pulmonary adenocarcinomas and in adenocarcinoma cell lines of 
the lung. SP-A has been detected mostly in bronchioalveolar carcinomas 
(Dempo, et al., Path. Res. Pract., Vol. 182, pg. 669 (1987); Kitinya, Acta 
Pathol. Japan, Vol. 36, pg. 127 (1986); Singh, et al., Am. J. Path., Vol. 
102, pg. 195 (1981); Espinoza, et al., Cancer, Vol. 54, pg. 2182 (1984)), 
which accounts for only about 2% of all primary carcinomas and examples of 
this tumor type were not available in the present study. The number of 
studies disclosing information on the immunohistochemical profile of SP-A 
on other types of lung carcinomas and malignancies arising in other body 
sites is small (Singh, et al., 1981; Mizutani, et al., Cancer, Vol. 61, 
pg. 532 (1988)). SP-A is not expressed in non-pulmonary tissues in the 
human as assessed by in situ hybridization or immunohistochemistry 
(Floros, et al., J. Biol. Chem., Vol. 261, pg. 828 (1986)). Staining for 
SP-A, however, was also detected in the breast tumors in the present 
study, but the tinctorial quality and the distribution of intracellular 
staining of SP-A were distinct in the breast tumors compared to the lung 
tumors, raising the possibility that the immunostaining for SP-A in breast 
carcinoma represents cross reactivity with other cellular proteins. A 
close relationship of the structure of SP-A to a number of cellular 
proteins may contribute to lack of specificity of the SP-A antiserum 
observed in the three breast tumors in the present study and the presence 
of trace amounts of reactivity also described in rare carcinomas of the 
thyroid gland (Shimosato, et al., Lung Cancer Differentiation: 
Implications for Diagnosis and Treatment, Bernal, et al., eds., New York, 
Marcel Dekker, Inc., pgs. 275 (1992)) and breast (Linnoila, et al., Am. J. 
Clin. Pathol., Vol. 97, pg. 233 (1992)). In contrast, staining for SP-B 
was entirely specific for lung carcinomas. Like SP-A, SP-B is expressed 
only in respiratory epithelial cells as assessed by in situ hybridization 
and immunostaining in a pattern similar to that of SP-A (Stahlman, et al., 
1992). Specificity of staining of adenocarcinoma for SP-B supports its 
utility as a marker and diagnosis of pulmonary adenocarcinoma. 
The present example was designed to test the applicability of 
immunostaining for antisera generated against SP-A, SP-B and TTF-1 for 
routine use for assessment of surgical specimens. Antibody staining 
procedures utilized in the present example were useful for routine 
pathological analysis of bronchial biopsies and surgical pathologic 
specimens. The use of these relatively reliable cell markers in routine 
pathological specimens, may help to distinguish adenocarcinomas of the 
lung from those arising in other tissues, such as, for example, the 
breast. The presence of TTF-1 in both non-small cell and small cell 
carcinomas of the lung supports the theory of a common histogenesis for 
both groups of malignancies. 
Example 7 
Gene sequence of human TTF-1 protein 
Reagents, Bacterial Strains, and Plasmids--Restriction endonucleases and 
enzymes used in cloning reactions were purchased from Life Technologies, 
Inc. A random primer kit (Stratagene) was used to radio-label cDNA 
fragments with [.alpha..sup.32 P] dCTP. Oligonucleotides were labeled with 
[.gamma..sup.32 P] ATP by kinase reaction. Radioisotopes were purchased 
from DuPont NEN. Escherichia coli DH5.alpha. or DH5.alpha.F.sup.1 was used 
as a host strain for pUC and pBluescript plasmids and M13 phage. 
Identification of Genomic Clone--A human cosmid (pWE15, Stratagene) genomic 
library was kindly provided by Dr. A. Menon (University of Cincinnati 
College of Medicine) and screened using a 1.3-kb rat TTF-1 cDNA clone, a 
gift from Dr. R. Di Lauro (Stazione Zoologica "Anton Dohrn," Naples, 
Italy). Hybridization was performed at 60.degree. C. under conditions 
recommended for Hybond (Amersham Corp.). The final wash was in 
0.2.times.SSC (1.times.SSC, pH 7.0:150 mM NaCl, 15 mM sodium citrate) at 
65.degree. C. Positive colonies were screened at lower density an 
additional three times to achieve colony purity. Filters were exposed to 
Kodak XAR film at -80.degree. C. for 2 nights. Three genomic equivalents 
were screened in duplicate, and two positive clones were identified. 
Initial restriction analyses of the two clones were identical, so one 
clone was selected for more detailed analysis. 
Southern Blot Analysis--DNA from human lung adenocarcinoma line H441-4 and 
from the cosmid clone was digested with BamiHI, EcoRI, HindIII, and KpnI, 
electrophoresed through an agarose gel, transferred to Hybond (Amersham), 
and probed with the labeled rat TTF-1 cDNA. Filters were washed at a final 
stringency of 0.2.times.saline/sodium phosphate/EDTA, 0.1% SDS at 
65.degree. C. and exposed to Kodak XAR film at -80.degree. C. In addition, 
the cosmid clone DNA was digested with additional restriction enzymes, 
subjected to Southern analysis, and probed under less stringent conditions 
with labeled oligonucleotide probes made to various regions of the rat 
TTF-1 cDNA. 
DNA Sequence Analysis--A 5.7-kb Xhol-HindIII fragment and a 4.6-kb BamHI 
fragment containing the human TTF-1 gene were subcloned into pUC18 and -19 
and into M13 mp 18 and 19. The TTF-1 gene was sequenced using the U.S. 
Biochemical Corp. sequenase kit, using either single-stranded or 
double-stranded DNA. Human TTF-1 specific oligonucleotides were 
synthesized and used as primers as the sequence was generated. The 
resulting DNA sequence was stored and analyzed on a MacIntosh IIs, using 
the program DNA Star. 
RNA Extraction and Northern Analysis--Cell lines were maintained in 
standard tissue culture prior to harvest including HeLa cervical 
epithelial cells, 3T3 fibroblasts, A549, H441, H820, 9/HTEo-, and BEAS-2B 
pulmonary adenocarcinomas, H441 and H345 small cell carcinomas were 
obtained from ATCC and maintained as suggested prior to harvest. Total RNA 
was isolated by an adapted method of Chirgwin et al., Biochemistry, Vol. 
18, pgs. 5294-5299 (1979). Tissue was homogenized in 4M guanidine 
thiocyanate, 0.5% N-lauroylsarcosine, 25 mM sodium citrate, and 0.1 M 
.beta.-mercaptoethanol. Cells grown in culture were lysed directly on the 
plate using the same buffer. Thereafter, Phase Lock gels (5 Prime.fwdarw.3 
Prime, Inc., Boulder, Colo.) were used to prepare RNA. RNA quantity was 
determined by absorbance at 260 nm. 
Total RNA (20 .mu.g) was electrophoresed through a 1.0% agarose, 7% 
formaldehyde gel, transferred to Hybond (Amersham) or Nytran (Schleicher & 
Schuell), and bound to the filter by UV cross-linking. Filters were 
hybridized overnight at 42.degree. C. in 50% formaldehyde plus standard 
sodium phosphate-EDTA solution as recommended, using .sup.32 P-random 
primer-labeled rat TTF-1 cDNA as probe. Filters were washed to a final 
stringency of 0.2.times.saline/sodium/phosphate/EDTA, 0.1% SDS at 
60.degree. C. and exposed to Kodak XAR-2 film. 
Luciferase Assays--The pGL2 vector, a luciferase reporter vector, was 
purchased from Promega. Two human TTF-1 gene fragments, HindIII/SspI and 
SmaI/SspI, were cloned into the multiple cloning site of the pGL2 basic 
construct to generate pGL2-2.7 kb and pGL2-0.55 kb, respectively, as seen 
in FIG. 38B. 
Human NCI-H441-4 (H441) and mouse MLE-15 cells were maintained as described 
previously (Bohinski et al., 1994; Wikenheiser et al., 1993). NIH-3T3 
cells (3T3) were maintained in Dulbecco's modified Eagle's medium 
containing 10% heat-inactivated bovine serum. Transfections were performed 
by the calcium phosphate co-precipitation method as described by 
Rosenthal, Methods Enzymol. Vol. 452, pgs. 704-720 (1987), except that 
glycerol shock was not used. Luciferase reporter plasmid (5 pmol) and 2.5 
pmol of the internal control plasmid, pCMV-.beta.gal (MacGregor et al., 
Methods Mol. Biol., Vol. 7, pgs. 1-9 (1989)) were co-transfected. Cells 
were incubated for approximately 18 hrs., washed once with Hanks' balanced 
salt solution (Life Technologies, Inc.), and returned to culture in 
original media for an additional 24 hrs. for MLE-15 cells, 72 hrs. for 
H441 cells, and 48 hrs. for 3T3 cells. Cells were harvested with reporter 
lysis buffer (Promega) followed by a rapid single freeze-thaw cycle. The 
lysates were prepared, and aliquots were assayed for .beta.-galactosidase 
activity (Bohinski et al., 1994) and for luciferase activity using a 
luminometer (Analytical Luminescence Laboratory, San Diego, Calif.). To 
correct for variations in transfection efficiency, assays were normalized 
to .beta.-galactosidase activity. 
Immunohistochemical Localization of Human TTF-1--Immunohistochemistry was 
performed on post-mortem samples of formalin-fixed tissues of human fetal 
and neonatal or adult lung obtained under protocols approved by the Human 
Research Committee, Vanderbilt University, Nashville, Tenn. 
Immunoperoxidase methods using a streptavidin-biotin kit (Biogenex) or an 
avidin biotin kit (Vectastain Elite ABC kit, Vector Laboratories) were 
used for immunolocalization of the antigen (Sternberger, ed., 
Immunocytochemistry, 2nd Ed., pgs. 104-114, John Wiley & Sons, Inc., New 
York (1979)). Antigen retrieval systems, using microwave heating, markedly 
enhanced TTF-1 staining and were routinely used. Anti-rat TTF-1 serum, 
produced in rabbits, was kindly provided by Dr. R. Di Lauro and used at a 
dilution of 1:1000 to 1:2000. Specificity was established by replacing the 
specific TTF-1 antibody with nonimmune rabbit antisera. Staining was 
completely blocked by preadsorption of the antisera with recombinant TTF-1 
(data not shown). Sections were counterstained with hematoxylin or nuclear 
fast red prior to photography. The staining represents data from more than 
20 distinct samples obtained at post-mortem at ages 11 weeks of gestation 
through adulthood. 
Cloning and Nucleotide Sequence Analysis of the Human TTF-1 Gene--Two 
identical genomic TTF-1 clones were isolated from an amplified human 
genomic library by hybridization screening with the rat TTF-1 cDNA under 
stringent conditions. Restriction fragment analysis of the cosmid clone 
was similar to that of DNA from human adenocarcinoma cell line H441 (FIG. 
37), indicating the presence of only one human TTF-1 gene. 
As shown in FIG. 37, 20 .mu.g of DNA from the cosmid clone (FIG. 37A) or 
from H441 cells (FIG. 37B) was digested with BamHI (lane 1), EcoRI (lane 
2), HindIII (lane 3), or KpnI (lane 4), and subjected to Southern analysis 
using the rat TTF-1 cDNA as a probe. 
The TTF-1 locus was contained within a 4.6-kb BamHI fragment consisting of 
two exons and one intron (FIG. 38A). The predicted human TTF-1 peptide of 
371 amino acids shared close identity with the amino acid sequence 
predicted by the rat TTF-1 cDNA sequence and 92.4% identity with the 
nucleotide sequence of the rat TTF-1 cDNA. The human TTF-1 gene consisted 
of two exons interrupted by a single exon of approximately 1 kb flanked by 
consensus splice donor acceptor sites that fit splice-acceptor donor 
rules. The restriction map, location of the exons, and nucleotide sequence 
are provided in FIGS. 38A and 39. The cosmid clone included the 
transcriptional start site previously identified for rat TTF-1 and 
termination signals consistent with the size of the 2.3-kb mRNA detected 
by Northern blot analysis of RNA from rat lung tissue (data not shown) and 
mouse and human pulmonary adenocarcinoma cells (H441) (FIG. 40). 
FIG. 40A is the Northern blot analysis of 20 .mu.g of total RNA from MLE-15 
(lane 1), MLE-F6 (lane 2), 3T3 (lane 3), and H441 cells (lane 4). The 
probe employed was the rat TTF-1 cDNA. FIG. 40B is the Northern blot 
analysis of 15 .mu.g of total RNA from human cell lines HeLa (lane 1), 
H441 (lane 2), H345 (lane 3), H446 (lane 4), BEAS-2B (lane 5), 9/HTE.sub.o 
- (lane 6), and A549 (lane 7). The probe used was a SacII-Sau 3AI fragment 
of rat TTF-1 cDNA. 
TTF-1 mRNA was detected in human pulmonary adenocarcinoma cells H441 and 
H820 (data not shown) and small cell carcinoma H345 but was not detected 
in 9/HTE.sub.o - or BEAS-2B (tracheal-bronchial epithelial cell lines), 
A549, HeLa, or 3T3 cells, demonstrating the cell selectivity of TTF-1 
expression. The size of TTF-1 mRNA was similar to that previously 
described in the rat thyroid and thyroid carcinoma cells (Guazzi, et al., 
EMBO, J., Vol. 9, pgs. 3631-3639 (1990)). The start of transcription was 
mapped by S1 analysis of mRNA from MLE-15 and H441 cells demonstrating 
three closely apposed transcriptional start sites located approximately 
-196 base pairs from the ATG initiator methionine in both species (data 
not shown). 
Transcriptional Activity of the 5'-Region of the TTF-1 Gene--Genomic 
fragments of 2.7 and 0.55 kb of the 5'-region of the TTF-1 gene were 
ligated into a firefly luciferase plasmid and transfected into H441, 
MLE-15, and 3T3 fibroblast cell lines. The TTF-1 luciferase constructs 
expressed luciferase activity in pulmonary adenocarcinoma cells H441 and 
MLE-15; activity of these constructs was detected, albeit at lower levels, 
in 3T3 cells (FIG. 41). 
The cells were co-transfected with a CMV-.beta.gal construct as hereinabove 
described, and results are plotted as units of luciferase activity per 
unit of .beta.-galactosidase and represent at least three separate 
experiments performed in quadruplicate. 
Activity of the TTF-1-luciferase constructs was approximately 10-20-fold 
higher in mouse lung epithelial cells (MLE-15) and H441- 4 cells than in 
3T3 cells. Luciferase activity was higher in the 2.7-kb TTF-1-luciferase 
construct than in the 0.55-kb TTF-1-luciferase constructs in all cell 
types. 
Distribution of TTF-1 in the Developing Human Lung--TTF-1 was detected by 
immunohistochemistry in nuclei of the respiratory epithelium in human 
fetal lung as early as 11-12 weeks of gestation. Immunostaining was 
observed in the developing airways in a distribution pattern similar to 
that previously described for pro-SP-C (Khoor et al., J. Histochem. 
Cytochem., Vol. 42, pgs. 1187-1199 (1994)) (FIG. 42). FIG. 42 depicts 
immunoperoxidase staining to stain human lung samples from 12 weeks of 
gestation (FIGS. 42A and 42B), 37 weeks of gestation (FIGS. 42C and 42D), 
and adult (FIGS. 42E and 42F). FIG. 42F is a control slide of adult lung 
tissue without primary antibody. The slides were counterstained with 
hematoxylin (FIGS. 42A, 42B, 42C, and 42D) or nuclear fast red (FIGS. 42E 
and 42F). Magnification of FIGS. 42A, 42B and 42C is 530.times., and 
magnification of FIGS. 42D, 42E, and 42F is 425.times.. 
TTF-1 was detected in subsets of respiratory epithelial cells in the 
developing lung, including nonciliated bronchiolar, and rarely in 
nonciliated bronchila respiratory epithelial cells in the immature lung 
(FIG. 42). At the time of birth, TTF-1 was detected in alveolar Type II 
epithelial cells and in subsets of nonciliated bronchiolar epithelial 
cells. TTF-1 was not detected in alveolar Type I cells or ciliated 
epithelial cells. The distribution of cells expressing TTF-1 is consistent 
with the overlapping distribution patterns of surfactant proteins A, B, 
and C and CCSP (Khoor et al., J. Histochem. Cytochem., Vol. 41, pgs. 
1311-1319 (1993); Khoor et al., 1994; Singh et al., J. Histochem. 
Cytochem., Vol. 36, pgs. 73-80 (1988)). In the adult lung, TTF-1 was 
detected readily in subsets of nonciliated bronchiolar epithelial cells 
and was most prominent in Type II epithelial cells but was excluded from 
Type I cells (FIG. 42). 
The disclosures of all patents, publications (including published patent 
applications), database accession numbers, and depository accession 
numbers referenced in this specification are specifically incorporated 
herein by reference in their entirety to the same extent as if each such 
individual patent, publication, and database accession number, and 
depository accession number were specifically and individually indicated 
to be incorporated by reference. 
It is to be understood, however, that the scope of the present invention is 
not to be limited to the specific embodiments described above. The 
invention may be practiced other than as particularly described and still 
be within the scope of the accompanying claims. 
__________________________________________________________________________ 
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(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 4: 
- - GCCAAG - # - # - 
# 6 
- - - - (2) INFORMATION FOR SEQ ID NO: 5: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 5: 
- - CTCAAG - # - # - 
# 6 
- - - - (2) INFORMATION FOR SEQ ID NO: 6: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 6: 
- - CTCCAG - # - # - 
# 6 
- - - - (2) INFORMATION FOR SEQ ID NO: 7: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 7: 
- - GTCAAG - # - # - 
# 6 
- - - - (2) INFORMATION FOR SEQ ID NO: 8: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8: 
- - TCTAAG - # - # - 
# 6 
- - - - (2) INFORMATION FOR SEQ ID NO: 9: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9: 
- - GTTAAG - # - # - 
# 6 
- - - - (2) INFORMATION FOR SEQ ID NO: 10: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10: 
- - CTGAAG - # - # - 
# 6 
- - - - (2) INFORMATION FOR SEQ ID NO: 11: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11: 
- - TCCAGG - # - # - 
# 6 
- - - - (2) INFORMATION FOR SEQ ID NO: 12: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #12: 
- - CCGAAC - # - # - 
# 6 
- - - - (2) INFORMATION FOR SEQ ID NO: 13: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #13: 
- - CCCAAG - # - # - 
# 6 
- - - - (2) INFORMATION FOR SEQ ID NO: 14: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #14: 
- - CATAAG - # - # - 
# 6 
- - - - (2) INFORMATION FOR SEQ ID NO: 15: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 6 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #15: 
- - TAGAGA - # - # - 
# 6 
- - - - (2) INFORMATION FOR SEQ ID NO: 16: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 16: 
- - TCAAGCACCT GGAGGGCTCT - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO: 17: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 17: 
- - GGAGGGCTCT TCAGAGCAAA - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO: 18: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 18: 
- - AGGTGCCACT CATAGAAAGC - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO: 19: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 19: 
- - TTGTTTCTGC CAAGTGCTGG - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO: 20: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 20: 
- - GATGCCCACT CAAGCTTAGA - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO: 21: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 21: 
- - GGTGACCACT CCAGGACATG - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO: 22: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 22: 
- - ACTGATTACT CAAGTATTCT - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO: 23: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 23: 
- - GGAGCAGACT CAAGTAGAGG - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO: 24: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 24: 
- - ACTGCCCAGT CAAGTGTTCT - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO: 25: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 25: 
- - AGCACCTGGA GGGCTCTTCA GAGC - # - # 
24 
- - - - (2) INFORMATION FOR SEQ ID NO: 26: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (ix) FEATURE: 
(D) OTHER INFORMATION: - # V is adenine, cytosine, or 
guanine; - #W is adenine, thymine, or uracil; R 
is adenin - #e or guanine; K is guanine, thymine, 
or uracil - #. 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 26: 
- - VAWTRTTKRW TW - # - # 
- # 12 
- - - - (2) INFORMATION FOR SEQ ID NO: 27: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 27: 
- - CAGTGTTTGC CT - # - # 
- # 12 
- - - - (2) INFORMATION FOR SEQ ID NO: 28: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 19 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 28: 
- - GCAAAGACAA ACACTGAGG - # - # 
- # 19 
- - - - (2) INFORMATION FOR SEQ ID NO: 29: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (ix) FEATURE: 
(A) NAME/KEY: PCR p - #rimer 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 29: 
- - CAGGAACATG GGAGTCTGGG - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO: 30: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 20 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (ix) FEATURE: 
(A) NAME/KEY: PCR p - #rimer 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 30: 
- - CAGTGCCTGG GCCACAGAGC - # - # 
- # 20 
- - - - (2) INFORMATION FOR SEQ ID NO: 31: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 31: 
- - TGTTTGT - # - # - 
# 7 
- - - - (2) INFORMATION FOR SEQ ID NO: 32: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 32: 
- - TGAGTCA - # - # - 
# 7 
- - - - (2) INFORMATION FOR SEQ ID NO: 33: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 33: 
- - TGGAGGGCTC T - # - # 
- # 11 
- - - - (2) INFORMATION FOR SEQ ID NO: 34: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 12 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 34: 
- - CAAACACTGA GG - # - # 
- # 12 
- - - - (2) INFORMATION FOR SEQ ID NO: 35: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 7 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - # 35: 
- - TGTTTGC - # - # - 
# 7 
- - - - (2) INFORMATION FOR SEQ ID NO: 36: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 46 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (ix) FEATURE: 
(A) NAME/KEY: PCR p - #rimer 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 36: 
- - TGGACAGGCG CGCCCGGCAC TTACCCTGCG TCAAGAGCCA GGAAGG - # 
46 
- - - - (2) INFORMATION FOR SEQ ID NO: 37: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (ix) FEATURE: 
(A) NAME/KEY: PCR p - #rimer 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 37: 
- - CGTCATGGCC ATATGGGCCT AGCCACTGCA GTAGGTGCGA CTTGGCCATG G - # 
51 
- - - - (2) INFORMATION FOR SEQ ID NO: 38: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 46 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (ix) FEATURE: 
(A) NAME/KEY: PCR p - #rimer 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 38: 
- - TGGACAGGCG CGCCCAGGGC TTGCCCTGGG TTAAGAGCCA GGCAGG - # 
46 
- - - - (2) INFORMATION FOR SEQ ID NO: 39: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 51 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (ix) FEATURE: 
(A) NAME/KEY: PCR p - #rimer 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 39: 
- - CGTCATGGCC ATATGGGCCC AGCCACTGCA GCAGGTGTGA CTCAGCCATG G - # 
51 
- - - - (2) INFORMATION FOR SEQ ID NO: 40: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #40: 
- - CGCACGCGTG AACATGGGAG TCTGGGCAGG - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO: 41: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #41: 
- - CGCACGCGTC AGAAGATTTT TCCAGGGGAA - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO: 42: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #42: 
- - GCGCTCGAGC CACTGCAGCA GGTGTGACTC - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO: 43: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #43: 
- - CGCACGCGTC AGGGCTTGCC CTGGGTTAAG - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO: 44: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #44: 
- - GCGCTCGAGG CCTGGGTGTT CCCCTCCCAT - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO: 45: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #45: 
- - CGCACGCGTG CCTGGGTGTT CCCCTCCCAT - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO: 46: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 bases 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #46: 
- - GCGCTCGAGC AGGGCTTGCC CTGGGTTAAG - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO: 47: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #47: 
- - CAGGGCTTGC CCTGGGTTAA G - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO: 48: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 21 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #48: 
- - GCCTGGGTGT TCCCCTCCCA T - # - # 
- #21 
- - - - (2) INFORMATION FOR SEQ ID NO: 49: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 192 bas - #es 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: genomic DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 49: 
- - CTGGGAAAAG GTGGGATCAA GCACCTGGAG GGCTCTTCAG AGCAAAGACA AA - 
#CACTGAGG 60 
- - TCGCTGCCAC TCCTACAGAG CCCCCACGCC CCGCCCAGCT ATAAGGGGCC AT - 
#GCCCCAAG 120 
- - CAGGGTACCC AGGCTGCAGA GGTGCCATGG CTGAGTCACA CCTGCTGCAG TG - 
#GCTGCTGC 180 
- - TGCTGCTGCC CA - # - # 
- # 192 
- - - - (2) INFORMATION FOR SEQ ID NO: 50: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 53 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: genomic DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 50: 
- - TGAGAAGACC TGGAGGGCTC TCAAGACACA GGCAAACACT GAGGTCAGCC TG - #T 
53 
- - - - (2) INFORMATION FOR SEQ ID NO: 51: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 55 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: genomic DNA 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 51: 
- - GATCAAGCAC CTGGAGGGCT CTTCAGAGCA AAGACAAACA CTGAGGTCGC TG - #CCA 
55 
- - - - (2) INFORMATION FOR SEQ ID NO: 52: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 52: 
- - ACGCAGGACT TGTTTGTTCT AG - # - # 
22 
- - - - (2) INFORMATION FOR SEQ ID NO: 53: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 22 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 53: 
- - CGACCTCAGT GTTTGTCTTT GC - # - # 
22 
- - - - (2) INFORMATION FOR SEQ ID NO: 54: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 54: 
- - TCTGATTATT GACTTAGTCA AGCG - # - # 
24 
- - - - (2) INFORMATION FOR SEQ ID NO: 55: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 55: 
- - AGCACCTGGA GGGCTCTTCA GAGC - # - # 
24 
- - - - (2) INFORMATION FOR SEQ ID NO: 56: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 56: 
- - ATCAAGCACC TGGAGGGC - # - # 
- # 18 
- - - - (2) INFORMATION FOR SEQ ID NO: 57: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 57: 
- - GGGCTCTTCA GAGCAAAG - # - # 
- # 18 
- - - - (2) INFORMATION FOR SEQ ID NO: 58: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 58: 
- - GCCCTCCAGG TGCTTGAT - # - # 
- # 18 
- - - - (2) INFORMATION FOR SEQ ID NO: 59: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 18 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 59: 
- - GGGCTCTTCA GAGCAAAG - # - # 
- # 18 
- - - - (2) INFORMATION FOR SEQ ID NO: 60: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 16 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (ix) FEATURE: 
(D) OTHER INFORMATION: - # N is a nucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 60: 
- - GCNCTNCAGN NNNNNG - # - # 
- # 16 
- - - - (2) INFORMATION FOR SEQ ID NO: 61: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 11 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (ix) FEATURE: 
(D) OTHER INFORMATION: - # N is a nucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 61: 
- - GNNCACTCAA G - # - # 
- # 11 
- - - - (2) INFORMATION FOR SEQ ID NO: 62: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 62: 
- - CACTGCCCAG TCAAGTGTTC TTGA - # - # 
24 
- - - - (2) INFORMATION FOR SEQ ID NO: 63: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 50 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 63: 
- - GCCACCCTCA AGGTTCTAAG TGCTCTTCTT GTTAAGTGCT CTGAAGGAAC - # 
50 
- - - - (2) INFORMATION FOR SEQ ID NO: 64: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 64: 
- - TCTAAGTGCT CTTCTTGTTA AGTGCTCTGA - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO: 65: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 24 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 65: 
- - GTGCCACCCT CAAGGTTCTA AGTG - # - # 
24 
- - - - (2) INFORMATION FOR SEQ ID NO: 66: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 23 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 66: 
- - GTTAAGTGCT CTGAAGGAAC CTG - # - # 
23 
- - - - (2) INFORMATION FOR SEQ ID NO: 67: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 14 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 67: 
- - TCTAAGTGCT CTTC - # - # 
- # 14 
- - - - (2) INFORMATION FOR SEQ ID NO: 68: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 109 bas - #es 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 68: 
- - CAGGGCTTGC CCTGGGTTAA GAGCCAGGCA GGAAGCTCTC AAGAGCATTG CT - #CA 
60 
- - GAGGGGGCCT GGGTGGCCCA GGGAGGGGAT GCGAGGGGAA CACCCAGGC - # 
109 
- - (2) INFORMATION FOR SEQ ID NO: 69: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 69: 
- - CAGGGCTTGC CCTGGGTTAA GAGCCAGGCA - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO: 70: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 70: 
- - TAGGGGGATC CCTGGGTTAA GAGCTAGGCA - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO: 71: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 71: 
- - GCCAGGCAGG AAGCTCTCAA GAGCATTG - # - # 
28 
- - - - (2) INFORMATION FOR SEQ ID NO: 72: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 28 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 72: 
- - GCCAGGTAGG AAGCTCTATC CAGCATTG - # - # 
28 
- - - - (2) INFORMATION FOR SEQ ID NO: 73: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 73: 
- - AGCATTGCTC AAGAGTAGAG GGGGCCTGGG - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO: 74: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 30 base - #s 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 74: 
- - AGCATTGCTA TCCAGTAGAG GGGGCCTGGG - # - # 
30 
- - - - (2) INFORMATION FOR SEQ ID NO: 75: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 3293 ba - #ses 
(B) TYPE: nucleic a - #cid 
(C) STRANDEDNESS: sing - #le 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: oligonucleotide 
- - (ix) FEATURE: 
(A) NAME/KEY: human - #TTF-1 gene 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 75: 
- - AACTTAAAGG TGTTTACCTT GTCATCAGCA TGTAAGCTAA TTATCTCGGG CA - 
#AGATGTAG 60 
- - GCTTCTATTG TCTTGTTGCT TTAGCGCTTA CGCCCCGCCT CTGGTGGCTG CC - 
#TAAAACCT 120 
- - GGCGCCGGGC TAAAACAAAC GCGAGGCAGC CCCCGAGCCT CCACTCAAGC CA - 
#ATTAAGGA 180 
- - GGACTCGGTC CACTCCGTTA CGTGTACATC CAACAAGATC GGCGTTAAGG TA - 
#ACACCAGA 240 
- - ATATTTGGCA AAGGGAGAAA AAAAAAGCAG CGAGCTTCGC CTTCCCCCTC TC - 
#CCTTTTTT 300 
- - TTCCTCCTCT TCCTTCCTCC TCCAGCCGCC GCCGAATCAT GTCGATGAGT CC - 
#AAAGCACA 360 
- - CGACTCCGTT CTCAGTGTCT GACATCTTGA GTCCCCTGGA GGAAAGCTAC AA - 
#GAAAGTGG 420 
- - GCATGGAGGG CGGCGGCCTC GGGGCTCCGC TGGCGGCGTA CAGGCAGGGC CA - 
#GGCGGCAC 480 
- - CGCCAACAGC GGCCATGCAG CAGCACGCCG TGGGGCACCA CGGCGCCGTC AC - 
#CGCCGCCT 540 
- - ACCACATGAC GGCGGCGGGG GTGCCCCAGC TCTCGCACTC CGCCGTGGGG GG - 
#CTACTGCA 600 
- - ACGGCAACCT GGGCAACATG AGCGAGCTGC CGCCGTACCA GGACACCATG AG - 
#GAACAGCG 660 
- - CCTCTGGCCC CGGATGGTAC GGCGCCAACC CAGACCCGCG CTTCCCCGCC AG - 
#TAAGTGAG 720 
- - GCCGCCCCAC TGCGGGGCCG CGGGCTGAGC TCAGGAGGTG CGGCGAGAGG CT - 
#CCAGAAGG 780 
- - CGCGGCGCCG GCAGGCTGCG CGCTGGGCAT CAGGGAGGGC GGCCCGGCAG CG - 
#GCGCCAGG 840 
- - GACTTGGGTG CGGGAGCTGG GGATGCTTCC CCCTGCTCGG CTGGGGGTCC AA - 
#GAACAGGC 900 
- - ACTTGGTAGC GCTGGGGTCC TGCGGTCAGA TGCGGGTACT CGGCGTCTCC TA - 
#GGCGCGGT 960 
- - GGACTGGCAG CTCTGCTCGG CGCAGAAGAC CTCGGGGAGC CAAGGGAAGC GA - 
#CCCCGAGC 1020 
- - TCAAGGAGCA GGGGCGAGCA GAGCGCGGAG AGGCTAGACC GGGCCAGGAG GG - 
#AGGCTGCC 1080 
- - CTGTTGGGAG GCACTCGAGC GCCCGGCCCG GCCCTCTCTC CAGCGGAGTC TG - 
#GGCAGGTG 1140 
- - GGAGGACTCG CAGTTCCAGA GGGGACTCTA AGGGTCCGAG CAGGTGCCCT CA - 
#CTGGGGCC 1200 
- - TGACAGGAGA GAAGCCAAGA GGCAAAGCGT CTGGGGGCTC CAGCTTTTGG AA - 
#GTCAACAC 1260 
- - CCCCTCTCCT AACCTCTCCA AACTGGGGTC TACCGTAGGA CCCCAGCTCC CG - 
#GCCTGAGC 1320 
- - CCAGTTCGCC GCCTGTGGCC AGCTAATCCT AATGCTCTGA CCCGGGCTGG GC - 
#ACGAAAGG 1380 
- - AGCAGAAGCG GCCTTTCCCC CACTGCGTCT TTTGGTTCGA AAGAGGGAAC TG - 
#AGACTGAG 1440 
- - GGAGGGCAGC CAGGGTTGGG GCTGTGAGCG CTCCAGTACA GCCCCCTCGA CG - 
#GTACGGCC 1500 
- - TGGGGCAGGC GCTGGCAGTT CCCCGCGGAT GGGCCTCTTG GGCCCCAGCG CT - 
#AGGCTGCC 1560 
- - TGGGTCAGGA GGGCGCCGTC GGTTGGGGCG GGCCGGGCGG GCCAATGGCG CG - 
#GAAAACAG 1620 
- - GGGTGGCCTG GCTCGGCCTG GCCCCGGCCG ACGCTGTGCG TTTGTCGCTT AC - 
#AGTCTCCC 1680 
- - GCTTCATGGG CCCGGCGAGC GGCATGAACA TGAGCGGCAT GGGCGGCCTG GG - 
#CTCGCTGG 1740 
- - GGGACGTGAG CAAGAACATG GCCCCGCTGC CAAGCGCGCC GCGCAGGAAG CG - 
#CCGGGTGC 1800 
- - TCTTCTCGCA GGCGCAGGTG TACGAGCTGG AGCGACGCTT CAAGCAACAG AA - 
#GTACCTGT 1860 
- - CGGCGCCGGA GCGCGAGCAC CTGGCCAGCA TGATCCACCT GACGCCCACG CA - 
#GGTCAAGA 1920 
- - TCTGGTTCCA GAACCACCGC TACAAAATGA AGCGCCAGGC CAAGGACAAG GC - 
#GGCGCAGC 1980 
- - AGCAACTGCA GCAGGACAGC GGCGGCGGCG GGGGCGGCGG GGGCACCGGG TG - 
#CCCGCAGC 2040 
- - AGCAACAGGC TCAGCAGCAG TCGCCGCGAC GCGTGGCGGT GCCGGTCCTG GT - 
#GAAAGACG 2100 
- - GCAAACCGTG CCAGGCGGGT GCCCCCGCGC CGGGCGCCGC CAGCCTACAA GG - 
#CCACGCGC 2160 
- - AGCAGCAGGC GCAGCACCAG GCGCAGGCCG CGCAGGCGGC GGCAGCGGCC AT - 
#CTCCGTGG 2220 
- - GCAGCGGTGG CGCCGGCCTT GGCGCACACC CGGGCCACCA GCCAGGCAGC GC - 
#AGGCCAGT 2280 
- - CTCCGGACCT GGCGCACCAC GCCGCCAGCC CCGCGGCGCT GCAGGGCCAG GT - 
#ATCCAGCC 2340 
- - TGTCCCACCT GAACTCCTCG GGCTCGGACT ACGGCACCAT GTCCTGCTCC AC - 
#CTTGCTAT 2400 
- - ACGGTCGGAC CTGGTGAGAG GACGCCGGGC CGGCCCTAGC CCAGCGCTCT GC - 
#CTCACGCT 2460 
- - TCCCTCCTGC CCGCCACACA GACCACCATC CACCGCTGCT CCACGCGCTT CG - 
#ACTTTTCT 2520 
- - TAACAACCTG GCCGCGTTTA GACCAAGGAA CAAAAAAACC ACAAAGGCCA AA - 
#CTGCTGGA 2580 
- - CGTCTTTCTT TCCCCCCCCC ACTCTAAAAT TTGTGGGTTT TTTTTTTTAA AA - 
#AAAAGAAA 2640 
- - ATGAAAAACA ACCAAGCGCA TCCAATCTCA AGGAATCTTT AAGCAGAGAA GG - 
#GCATAAAA 2700 
- - CAGCTTTGGG GGTGTCTTTT TTTGGTGATT CAAATGGGTT TTCCACGCTA GG - 
#GCGGGGCA 2760 
- - CAGATTGGAG AGGGCTCTGT GCTGACATGG CTCTGGACTC TAAAGACCAA AC - 
#TTCACTGT 2820 
- - GGGCACACTC TGCCAGCAAA GAGGACTCGC TTGTAAATAC CAGGATTTTT TT - 
#TTTTTTTT 2880 
- - TGAAGGGAGG ACGGGAGCTG GGGAGAGGAA AGAGTCTTCA ACATAACCCA CT - 
#TGTCACTG 2940 
- - ACACAAAGGA AGTGCCCCCT CCCCGGCACC CTCTGGCCGC CTAGGCTCAG CG - 
#GCGACCGC 3000 
- - CCTCCGCGAA AATAGTTTGT TTAATGTGAA CTTGTAGCTG TAAAACGCTG TC - 
#AAAAGTTG 3060 
- - GACTAAATGC CTAGTTTTTA GTAATCTGTA CATTTTGTTG TAAAAAGAAA AA - 
#CCACTCCC 3120 
- - AGTCCCCAGC CCTTCACATT TTTTATGGGC ATTGACAAAT CTGTGTATAT TA - 
#TTTGGCAG 3180 
- - TTTGGTATTT GCGGCGTCAG TCTTTTTCTG TTGTAACTTA TGTAGATATT TG - 
#GCTTAAAT 3240 
- - ATAGTTCCTA AGAAGCTTCT AATAAATTAT ACAAATTAAA AACGATTCTT TT - #T 
3293 
- - - - (2) INFORMATION FOR SEQ ID NO: 76: 
- - (i) SEQUENCE CHARACTERISTICS: 
(A) LENGTH: 371 ami - #no acids 
(B) TYPE: amino aci - #d 
(C) STRANDEDNESS: 
(D) TOPOLOGY: linear 
- - (ii) MOLECULE TYPE: protein 
- - (ix) FEATURE: 
(A) NAME/KEY: human - #thyroid transcription factor-1 
- - (xi) SEQUENCE DESCRIPTION: SEQ ID - #NO: 76: 
- - Met Ser Met Ser Pro Lys His Thr Thr Pro 
- #5 - #10 
- - Phe Ser Val Ser Asp Ile Leu Ser Pro Leu 
15 - # 20 
- - Glu Glu Ser Tyr Lys Lys Val Gly Met Glu 
25 - # 30 
- - Gly Gly Gly Leu Gly Ala Pro Leu Ala Ala 
35 - # 40 
- - Tyr Arg Gln Gly Gln Ala Ala Pro Pro Thr 
45 - # 50 
- - Ala Ala Met Gln Gln His Ala Val Gly His 
55 - # 60 
- - His Gly Ala Val Thr Ala Ala Tyr His Met 
65 - # 70 
- - Thr Ala Ala Gly Val Pro Gln Leu Ser His 
75 - # 80 
- - Ser Ala Val Gly Gly Tyr Cys Asn Gly Asn 
85 - # 90 
- - Leu Gly Asn Met Ser Glu Leu Pro Pro Tyr 
95 - # 100 
- - Gln Asp Thr Met Arg Asn Ser Ala Ser Gly 
105 - # 110 
- - Pro Gly Trp Tyr Gly Ala Asn Pro Asp Pro 
115 - # 120 
- - Arg Phe Pro Ala Ile Ser Arg Phe Met Gly 
125 - # 130 
- - Pro Ala Ser Gly Met Asn Met Ser Gly Met 
135 - # 140 
- - Gly Gly Leu Gly Ser Leu Gly Asp Val Ser 
145 - # 150 
- - Lys Asn Met Ala Pro Leu Pro Ser Ala Pro 
155 - # 160 
- - Arg Arg Lys Arg Arg Val Leu Phe Ser Gln 
165 - # 170 
- - Ala Gln Val Tyr Glu Leu Glu Arg Arg Phe 
175 - # 180 
- - Lys Gln Gln Lys Tyr Leu Ser Ala Pro Glu 
185 - # 190 
- - Arg Glu His Leu Ala Ser Met Ile His Leu 
195 - # 200 
- - Thr Pro Thr Gln Val Lys Ile Trp Phe Gln 
205 - # 210 
- - Asn His Arg Tyr Lys Met Lys Arg Gln Ala 
215 - # 220 
- - Lys Asp Lys Ala Ala Gln Gln Gln Leu Gln 
225 - # 230 
- - Gln Asp Ser Gly Gly Gly Gly Gly Gly Gly 
235 - # 240 
- - Gly Thr Gly Cys Pro Gln Gln Gln Gln Ala 
245 - # 250 
- - Gln Gln Gln Ser Pro Arg Arg Val Ala Val 
255 - # 260 
- - Pro Val Leu Val Lys Asp Gly Lys Pro Cys 
265 - # 270 
- - Gln Ala Gly Ala Pro Ala Pro Gly Ala Ala 
275 - # 280 
- - Ser Leu Gln Gly His Ala Gln Gln Gln Ala 
285 - # 290 
- - Gln His Gln Ala Gln Ala Ala Gln Ala Ala 
295 - # 300 
- - Ala Ala Ala Ile Ser Val Gly Ser Gly Gly 
305 - # 310 
- - Ala Gly Leu Gly Ala His Pro Gly His Gln 
315 - # 320 
- - Pro Gly Ser Ala Gly Gln Ser Pro Asp Leu 
325 - # 330 
- - Ala His His Ala Ala Ser Pro Ala Ala Leu 
335 - # 340 
- - Gln Gly Gln Val Ser Ser Leu Ser His Leu 
345 - # 350 
- - Asn Ser Ser Gly Ser Asp Tyr Gly Thr Met 
355 - # 360 
- - Ser Cys Ser Thr Leu Leu Tyr Gly Arg Thr 
365 - # 370 
- - Trp 
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