U.S. patent number 4,414,150 [Application Number 06/237,388] was granted by the patent office on 1983-11-08 for hybrid human leukocyte interferons.
This patent grant is currently assigned to Genentech, Inc.. Invention is credited to David V. Goeddel.
United States Patent |
4,414,150 |
Goeddel |
November 8, 1983 |
Hybrid human leukocyte interferons
Abstract
Disclosed herein are methods and means of microbially preparing
novel human hybrid leukocyte interferons, useful in the treatment
of viral and neoplastic diseases, by DNA recombination of parental
interferon genes, taking advantage of common restriction
endonuclease cleavage sites therein and in carrier expression
plasmids.
Inventors: |
Goeddel; David V. (Burlingame,
CA) |
Assignee: |
Genentech, Inc. (South San
Francisco, CA)
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Family
ID: |
26900564 |
Appl.
No.: |
06/237,388 |
Filed: |
February 23, 1981 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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205579 |
Nov 10, 1980 |
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Current U.S.
Class: |
530/351;
424/85.7; 435/320.1; 435/69.51; 435/811; 530/808; 930/142 |
Current CPC
Class: |
C07K
14/56 (20130101); C12N 15/71 (20130101); A61K
38/00 (20130101); Y10S 530/808 (20130101); Y10S
930/142 (20130101); Y10S 435/811 (20130101) |
Current International
Class: |
C07K
14/56 (20060101); C07K 14/435 (20060101); C12N
15/71 (20060101); A61K 38/00 (20060101); C07C
103/52 (); C07G 007/00 (); C12P 021/00 (); C12P
021/06 () |
Field of
Search: |
;424/85
;435/68,70,172,317,811 ;260/112R,112.5R |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Derynck et al., Nature 285, 542 (1980). .
Nagata et al., Nature 284, 316 (1980). .
Taniguchi et al., Proc. Japan. Acad. Sci. Ser B 55, 464 (1979).
.
Research Disclosure, Jul. 1979, pp. 351-352. .
Nienhuis et al., New Eng. J. Med. 297, 1318 (1977). .
Miozzari et al., Nature 277, 486 (1979). .
Itakura et al., Science 196, 1056 (1977)..
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Primary Examiner: Tanenholtz; Alvin E.
Assistant Examiner: Martinell; James
Attorney, Agent or Firm: Saxe; Jon S. Gould; George M.
Parent Case Text
This is a continuation in part of application Ser. No. 205,579,
filed Nov. 10, 1980.
Claims
What is claimed is:
1. An antiviral composition of matter comprising a polypeptide of
165-166 amino acids, optionally having an additional methionine
attached to the ordinarily first amino acid at the N-terminus, the
amino acid sequence of said polypeptide comprising, in sequence,
discrete sub-sequences corresponding in amino acid identity and
number to sub-sequences of different, naturally occurring leukocyte
interferons, the amino acid sequence of said polypeptide differing
from the amino acid sequence of naturally occurring leukocyte
interferons.
2. An antiviral composition of matter according to claim 1
comprising a polypeptide of 166 amino acids, the N-terminal portion
of said polypeptide consisting essentially of amino acids 1-92 of
LeIF-D and the carboxy terminal portion of said polypeptide
consisting essentially of amino acids 92-165 of LeIF-A, the carboxy
terminus of said polypeptide corresponding essentially to the
carboxy terminus of LeIF A.
3. An antiviral composition of matter according to claim 1
comprising a polypeptide of 165 amino acids, the N-terminal portion
of said polypeptide consisting essentially of amino acids 1-91 of
LeIF A and the carboxy terminal portion of said polypeptide
consisting essentially of amino acids 93-166 of LeIF-D, the carboxy
terminus of said polypeptide corresponding essentially to the
carboxy terminus of LeIF D.
4. An antiviral composition of matter according to claim 1
comprising a polypeptide of 166 amino acids, the N-terminal portion
of said polypeptide consisting essentially of amino acids 1-63 of
LeIF D and the carboxy terminal portion of said polypeptide
consisting essentially of amino acids 63-165 of LeIF A, the carboxy
terminus of said polypeptide corresponding essentially to the
carboxy terminus of LeIF A.
5. An antiviral composition of matter according to claim 1
comprising a polypeptide of 165 amino acids, the N-terminal portion
of said polypeptide consisting essentially of amino acids 1-62 of
LeIF A, the carboxy terminal portion of said polypeptide consisting
essentially of amino acids 64-166 of LeIF D, the carboxy terminus
of said polypeptide corresponding essentially to the carboxy
terminus of LeIF D.
6. An antiviral composition of matter according to claim 1
comprising a polypeptide of 165 amino acids, the N-terminal portion
of said polypeptide consisting essentially of amino acids 1-91 of
LeIF A and the carboxy terminal portion of said polypeptide
consisting essentially of amino acids 93-166 of LeIF B, the carboxy
terminus of said polypeptide corresponding essentially to the
carboxy terminus of LeIF B.
7. An antiviral composition of matter according to claim 1
comprising a polypeptide of 165 amino acids, the N-terminal portion
of said polypeptide consisting essentially of amino acids 1-91 of
LeIF A, the carboxy terminal portion of said polypeptide consisting
essentially of amino acids 93-166 of LeIF F, the carboxy terminus
of said polypeptide corresponding essentially to the carboxy
terminus of LeIF F.
Description
FIELD OF THE INVENTION
This invention relates to the microbial production, via recombinant
DNA technology, of hybrid leukocyte interferons for use in the
treatment of viral and neoplastic diseases, and to the means and
end products of such production.
CROSS REFERENCE TO RELATED APPLICATIONS
This application is in part related in subject matter to U.S.
patent applications of David V. Goeddel and Sidney Pestka, Ser. No.
205,578, filed Nov. 10, 1980 and Ser. No. 184,909 filed Sept. 8,
1980. The disclosure of each of the foregoing, commonly owned,
applications is incorporated herein by reference to illuminate the
background of the invention and to provide additional detail
relative to the practice thereof.
BACKGROUND OF THE INVENTION
Relatively homogeneous leukocyte interferons have been derived from
normal or leukemic donors' leukocytes. These interferons are a
family of proteins characterized by a potent ability to confer a
virus-resistant state in their target cells. In addition,
interferon can act to inhibit cell proliferation and modulate
immune response. These properties have prompted the clinical use of
interferon as a therapeutic agent for the treatment of viral
infections and malignancies.
More recently, recombinant DNA technology has been employed to
occasion the microbial production of a number of different
leukocyte interferons whose amino acid sequences exhibit on the
order of 70 percent homology, one relative to another, all as
disclosed in the aforementioned U.S. patent applications of Goeddel
an Pestka and in the manuscript of David V. Goeddel et al entitled
"The Structures of Eight Distinct Cloned Human Leukocyte Interferon
cDNAs", a copy of which is attached as Appendix A to the present
application and incorporated herein. The manner in which genes
encoding amino acid sequences of various leukocyte interferons
designated, inter alia, LeIF A,B,C,D,F,G and H, respectively, are
obtained from the cell line KG-1 described in Koeffler, H. P. and
Golde, D. W. Science 200, 1153-1154 (1978) is disclosed in the
aforementioned applications of Goeddel and Pestka. The cell line
KG-1 has been deposited with the American type culture collection,
ATCC Accession Number CRL 8031. Such genes, appropriately deployed
in plasmidic vehicles for bacterial expression, may be employed to
transform host bacteria, preferably E. coli K-12 strain 294,
American type culture collection accession No. 31446.
BRIEF SUMMARY OF THE INVENTION
Nucleotide sequence studies of genes encoding the various leukocyte
interferons reveals a degree of commonality amongst various of them
with regard to the presence and location of cleavage sites
recognized by particular restriction endonucleases. According to
the present invention, advantage may be taken of this commonality
to form, by DNA recombination, novel hydrid genes useful in the
microbial production of hybrid leukocyte interferons which may be
expected to exhibit in greater or lesser degree the antiviral and
other properties of interferons encoded by the parental genes. In
preferred embodiments of the invention, such hybrid leukocyte
interferons may exhibit enhanced activity relative to those encoded
by the parental genes.
The manner in which these and other objects and advantages of the
invention are obtained will become further apparent from the
detailed description which follows and from the accompanying
drawings in which:
FIG. 1 depicts nucleotide sequences of the coding regions of 8
leukocyte interferon ("LeIF") complementary DNA ("cDNA") clones. Of
these, one, correspondingly designated LeIF E, is an apparent
"pseudogene" encoding no active leukocyte interferon while another,
designated LeIF G, contains less than the full sequence for the
corresponding interferon species. The ATG translational initiation
codon and the termination triplet for each LeIF is underlined.
FIG. 2 depicts restriction endounuclease maps of eight types of
LeIF cloned cDNAs (A through H). Plasmids containing the clones
were constructed by the dC:dG tailing method, Goeddel, D.V. et al,
Nature 287, 411-416 (1980). Therefore the cDNA inserts can be
excised using Pst I, i.e., each end of each insert is a Pst I
restriction endonuclease cleavage site. The lines at the end of
each cDNA insert represent the flanking homopolymeric dC:dG tails.
The positions of Pvu II, Eco RI and Bgl II restriction sites are
indicated. Shaded regions of the Figure represent the coding
sequences of mature LeIFs; the cross-hatched regions indicate
signal peptide coding sequences; and the open regions show 3' and
5' noncoding sequences.
FIG. 3 is a comparision of the eight LeIF protein sequences
predicted from the nucleotide sequences. The one letter
abbreviations recommended by the IUPAC-IUB Commission on
Biochemical Nomenclature are used: A, alanine; C, cysteine; D,
aspartic acid; E, glutamic acid; F, phenylalanine; G, glycine; H,
histidine; I, isoleucine; K, lysine; L, leucine; M, methionine; N,
asparagine; P, proline; Q, glutamine; R, arginine; S, serine; T,
threonene; V, valine; W, tryptophan; and Y, tyrosine. The numbers
refer to amino acid position (S refers to signal piptide). The dash
in the 165 amino acid LeIF A sequence at position 44 is introduced
to align the LeIF A sequence with the 166 amino acid sequences of
the other LeIFs. The LeIF E sequence was determined by ignoring the
extra nucleotide (position 187 of FIG. 1) in its coding region. The
asterisks indicate in-phase termination codons. Amino acids common
to all LeIFs (excluding the pseudogene LeIF E) are also shown. The
underlined residues are amino acids which are also present in human
fibroblast interferon.
With reference to FIG. 1, nucleotides +1 to 69 correspond to S1 to
S23 amino acids of FIG. 3. Codon TGT (nucleotides 70 to 72) of FIG.
1 corresponds to cysteine (C, amino acid 1) of FIG. 3. In FIG. 3,
the Pvu II restriction endonuclease cleavage site occurs between
the codons for amino acids 92 and 93 in LeIF A,B,D,F and G, i.e.,
between nucleotides 346 and 347, of FIG. 1.
FIG. 4 compares the amino acid sequence of mature leukocyte
interferons A and D, a deletion of amino acid 44 in LeIF A being
indicated by dashes. Only those LeIF D amino acids which differ
from corresponding amino acids of LeIF A are depicted: the amino
acid sequence of LeIF D is otherwise identicial of LeIF A. FIG. 4
also indicates the relative position on the corresponding gene of
Bgl II and Pvu II restriction endonuclease cleavage sites employed
in forming preferred hybrid leukocyte genes of the invention.
FIGS. 5 and 6 illustrate the results of comparative testing of a
preferred hybrid leukocyte interferon of the invention ("LeIF-A/D")
for activity against encephalomyocarditis virus ("EMC") and
vesicular stomatitis virus ("VSV"), respectively in mouse
cells.
FIGS. 7 and 8 depict the results of comparative testing involving
LeIF-A/D and other interferons against EMC virus infections in,
respectively, mice and hamsters. The data in FIG. 7 result from
treatments i.p. 3 hrs. before infection. Dosages of LeIF-A/D and
LeIF-A are as titrated on WISH cells.
DESCRIPTION OF THE PREFERRED EMBODIMENTS
The LeIF hybrids are prepared, in accordance with the present
invention, by taking advantage of restriction endonuclease cleavage
sites commonly located within the individual parental genes and
each end thereof in conjunction with the same sites in carrier
expression plasmids (vectors). For example, the large (.about.3900
bp) fragment of a Xba I to Pst I digest of the pLeIF A trp 25
expression plasmid can be ligated with Xba I to Pvu II and Pvu II
to Pst I digest fragments of the various LeIF parental genes to
provide expression plasmids operable to obtain the corresponding
hybrod LeIF.
Each LeIF expression plasmid was constructed indipendently with
digest fragments isolated from the plasmids pLeIF A trp 25, pLeIF B
trp 7, pLeIF C trp 35, pLeIF D trp 11, pLeIF F trp 1, pLeIF G, and
pLeIF H, whose construction and description are described in the
aforementioned applications of Goeddel and Pestka, or pBR322 whose
construction and description is described by Bolivar et al., Gene
2, 95 (1977). In certain of these plasmids, "trp" designates a
tryptophan promoter-operator system most preferred for bacterial
expression, as described in the copending, commonly owned U.S.
patent application of Dennis G. Kleid et al., Ser. No. 133,296,
filed Mar. 24, 1980.
The methods and materials employed in the following constructions
were as in the aforementioned applications of Goeddel and Pestka.
The following Tble 1 provides the details for particular
constructions of hybrid LeIF plasmids hereof:
TABLE 1
__________________________________________________________________________
Total Expression Resultant LeIF Amino Plasmid Front Portion Back
Portion Expression Hybrid Acids* (Vector) Fragment Amino Acids
Fragment Amino Acids Plasmid
__________________________________________________________________________
AD 165 a XbaI-PvuII 1-91 PvuII-PstI 93-166 pLeIF AD of pLeIF A trp
25 of pLeIF D trp 11 trp (Pvu II) (285 bp) (.sup..about. 550 bp) DA
166 a XbaI-PvuII 1-92 PvuII-PstI 92-165 pLeIF DA of pLeIF D trp 11
of pLeIF A trp 25 trp (Pvu II) (288 bp) (.sup..about. 550 bp) AD
165 -- Bgl II-Pst I large 1-62 Bgl II-Pst I 64-166 pLeIF AD
fragment of pLeIF A of pLeIF D trp 11 trp (Bgl II) trp 25
(.sup..about. 600 bp) DA 166 -- Bgl II-Pst I large 1-63 Bgl II
partial- 63-165 pLeIF DA fragment pLeIF D Pst I of pLeIF A trp (Bgl
II) trp 11 trp 25 (.sup..about. 700 bp) AB 165 a XbaI-PvuII of LeIF
A 1-91 PvuII partial-PstI 93-166 pLeIF AB trp (Pvu II) trp 25 (285
bp) of pLeIF B trp 7 (.sup..about. 750 bp) AF 165 a XbaI-PvuII of
LeIF A 1-91 Pvu II Pst I 93-166 pLeIF AF trp (Pvu II) trp 25 (285
bp) of pLeIF F trp 1 (.sup..about. 700 bp) AG 165 a XbaI-PvuII of
LeIF A 1-91 PvuII-PstI 93-166 pLeIF AG trp (Pvu II) trp 25 (285 bp)
of pLeIF G (.sup..about. 750 bp) BA 166 b HindIII-PvuII of pLeIF
1-92 PvuII-PstI of 92-165 pLeIF BA trp (Pvu II) B trp 7
(.sup..about. 630 bp) pLeIF A trp 25 (.sup..about. 550 bp) BD 166 b
HindIII-PvuII of pLeIF 1-92 PvuII-PstI of 93-166 pLeIF BD trp (Pvu
II) B trp 7 (.sup..about. 630 bp) pLeIF D trp 11 (.sup..about. 550
bp) BF 166 b HindIII-PvuII of pLeIF 1-92 PvuII-PstI of 93-166 pLeIF
BF trp (Pvu II) B trp 7 (.sup..about. 630 bp) pLeIF F trp 1
(.sup..about. 700 bp) BG 166 b HindIII-PvuII of pLeIF 1-92
PvuII-PstI of 93-166 pLeIF BG trp (Pvu II) B trp 7 (.sup..about.
630 bp) pLeIF G (.sup..about. 750 bp) DB 166 a XbaI-PvuII of pLeIF
1-92 PvuII partial-PstI of 93-166 pLeIF DB trp (Pvu II) D trp 11
(288 bp) pLeIF B trp 7 (.sup..about. 750 bp) DF 166 a XbaI-PvuII of
pLeIF 1-92 PvuII-Pst I of 93-166 pLeIF DF trp (Pvu II) D trp 11
(288 bp) pLeIF F trp 1 (.sup..about. 700 bp) DG 166 a XbaI-PvuII of
pLeIF 1-92 PvuII-PstI of 93-166 pLeIF DG trp (Pvu II) D trp 11 (288
bp) pLeIF G (.sup..about. 750 bp) FA 166 a XbaI-PvuII of 1-92
PvuII-PstI of 92-165 pLeIF FA trp (Pvu II) pLeIF F trp 1 pLeIF A
trp 25 (288 bp) (.sup..about. 550 bp) FB 166 a XbaI-PvuII of 1-92
PvuII partial-PstI of 93-166 pLeIF FB trp (Pvu II) pLeIF F trp 1
pLeIF B trp 7 (288 bp) (.sup..about. 750 bp) FD 166 a XbaI-PvuII of
1-92 PvuII-PstI from 93-166 pLeIF FD trp (Pvu II) pLeIF F trp 1
pLeIF D trp 11 (288 bp) (.sup..about. 550 bp) FG 166 a XbaI-PvuII
of 1-92 PvuII-PstI from 93-166 pLeIF FG trp (Pvu II) pLeIF F trp 1
pLeIF G (288 bp) (.sup..about. 750 bp)
__________________________________________________________________________
*excluding N-- terminal methionine a large (.sup..about. 3900 bp)
fragment of Xba I to Pst I digest of pLeIF A trp 25. b large
(.sup..about. 3600 bp) fragment of Hind III to Pst I digest of
pBR322.
With further reference to Table I, the first four described hybrid
LeIFs have been produced from two LeIF expression plasmids. A Bgl
II site common to LeIF A and D cDNAs has been used to construct an
expression plasmid pLeIF trp AD (Bgl II) which codes for the 63
amino terminal amino acids of LeIF A and the 102 carboxy terminal
amino acids of LeIF D. The same site was utilized in the
construction of an expression plasmid pLeIF trp DA (Bgl II) which
codes for 64 amino terminal amino acids of LeIF D and 102 carboxy
terminal amino acids of LeIF A. The Pvu II site has been used in
the construction of two other hybrid interferon expression
plasmids: 91 amino terminal amino acids of A with 74 carboxy
terminal amino acids of D (LeIF tr AD (Pvu II)) and 92 amino
terminal amino acids of LeIF D with 74 carboxy terminal amino acids
of LeIF A (pLeIF trp DA (Pvu II)). In summary, for:
pLeIF AD trp (Pvu II): The large (.about.3900 bp) fragment of an
XbA I and Pst I digest of pLeIF A trp 25 was ligated with a 286 bp
Xba I-Pvu II fragment of pLeIF A trp 25 and an approximately 550 bp
Pvu II-Pst I fragment of pLeIF D trp 11;
pLeIF DA trp (Pvu II): The large (.about.3900 bp) fragment of an
Xba I and Pst I digest of pLeIF A trp 25 was ligated with a 288 bp
Xba I-Pvu-II fragment of pLeIF D trp 11 and an approximately 550 bp
Pvu II-Pst I fragment of pLeIF A trp 25;
pLeIF AD trp (Bgl II): The large fragment from a Bgl II, Pst I
digest of pLeIF A trp 25 was ligated with a .about.600 bp Bgl
II-Pst I fragment from pLeIF D trp 11; and
pLeIF DA trp (Bgl II): The large fragment from a Bgl II and Pst I
digest of pLeIF D trp 11 was ligated to an approximately 700 bp
fragment obtained by Pst I cleavage of pLeIF A trp 25 followed by
partial Bgl II digestion.
In the fifth depicted hybrid:
pLeIF AB trp (Pvu II): The large (.about.3900 bp) fragment of an
Xba I and Pst I digest of pLeIF A trp 25 was ligated with a 285 bp
Xba I-Pvu II fragment of pLeIF A trp 25 and an approximately 750 bp
Pvu II (partial)-Pst I fragment of pLeIF B trp 7.
In like manner, the other constructions depicted in Table I are so
defined. As a further example, in the construction of a LeIF C
and/or LeIF H portion containing hybrid, one can take advantage of
common Bbv I sites occurring at about nucleotide 294 (i.e., GCTGC)
of the gene sequences.
In like manner, plasmids suitable for the microbial expression of
other novel hybrid leukocyte interferons may be formed by
appropriate manipulation of double stranded DNA encoding all or
portions of the amino acid sequences of natural occurring leudocyte
interferons. Thus, a first double stranded DNA fragment is selected
which encodes the amino terminal of a first, naturally occurring
leukocyte interferon amino acid sequence and, proceeding therefrom
in the 3' direction, a substantial portion of the amino acid
sequence thereof. The fragment comprises a restriction endonuclease
cleavage site positioned adjacent codons for amino acid "n" of the
first leukocyte interferon, n amino acids constituting a
substantial portion of the amino acid sequence of the first
interferon. Cleavage with the restriction endonuclease yields a
fragment comprising the amino terminal of the first interfereon and
codons for approximately n amino acids. A second fragment
comprising all or a portion of the codons for the amino acid
sequence of a second, different leukocyte interferon is selected,
the fragment comprising a cleavage site for an identical
restriction endonuclease positioned adjacent codons for that amino
acid of the second interferon whose amino acid number (proceeding
from the amino terminal of the second interferon) is approximately
166-n. Cleavage of the second fragment with that restriction
endonuclease yields a product complementary to the "n" terminal
portion of the digestion product of the first fragment, such that
the digestion product of the second can be ligated to that of the
first, reforming the restriction endonuclease recognition site and
reconstituting the codon for amino acid n of the first interferon,
where lost in the initial digestion. The product of the restriction
endonuclease digestion of the second fragment preferably proceeds
from the end resulting from cleavage in the 3' direction through
nucleotides encoding the carboxy terminal of the second leukocyte
interferon.
Alternatively, hybrids containing substantial portions of the amino
acid sequences of more than two naturally occurring leukocyte
interferons may be formed, in which event, for example, the second
fragment mentioned above is additionally chosen to contain a second
restriction endonuclease site downstream from the first, the second
site being identical to a similarly positioned site within a
fragment encoding the carboxy terminal portion of a third leukocyte
interferon, etc. In the example referred to, the products of
successive restriction endonuclease operations may be
triple-ligated to form a hybrid gene encoding the amino terminal
portion of a first interferon, the mid-range amino acid sequence of
the second and the carboxy terminal portion of the third, (or, in
another variation of the first, where the first and third
interferons are the same).
Preferably, the first fragment mentioned above is derived from an
expression plasmid, i.e., one in which codons for the amino
terminal portion of the first leukocyte interferon are preceded by
an ATG or other translation initiation codon and a promoter or
promoter-operator system. As a result, the end product of the
manipulative operations described above will be a plasmid capable
of expressing the polypeptide encoded by the hybrid gene in
bacteria or other microbial organisms transformed with the plasmid.
Other means of configuring the hybrid gene for microbial expression
will be apparent to those skilled in the art.
In preferred embodiments of the invention, the hybrid genes encode
a novel leukocyte interferon amino acid sequence approximating
165-166 amino acids constituting a conjugate of substantial amino
acid sequences drawn from two or more different leukocyte
interferons selected from the group consisting of LeIF A, LeIF B,
LeIF C, LeIF D, LeIF E, LeIF F, LeIF G, and LeIF H as depicted in
FIG. 3. Most preferably, the novel leukocyte interferons encoded by
the hybrid genes comprise the amino acids specified and positioned
as indicated in the sequence "A11" of FIG. 3. The expression
products of plasmids formed according to the invention may be
tested for antiviral activity in conventional manner, as in the
biological activity determinations next described.
DEMONSTRATION OF ANTIVIRAL ACTIVITY
E. coli K-12 strain 294 was conventionally transformed with,
independently, the plasmids pLeIF trp A 25, pLeIF trp D, pLeIF trp
A/D (Bgl II) and pLeIF trp D/A (Bgl II). The transformants were
separately grown up in 5 ml cultures in L broth containing 5 mg/ml
tetracycline to an A.sub.550 of about 1.0, then diluted into one
liter of M9 media containing 5 .mu.g/ml tetracycline. Cells were
harvested when A.sub.550 reached 1.0 and cell pellets suspended in
10 ml of 15 percent sucrose, 50 mM tris-HCl (pH 8.0), 50 mM EDTA.
10 mg of lysozyme were added and, after 5 minutes at 0.degree. C.,
cells were disrupted by sonication. The samples were centrifuged 10
minutes at 15,000 rpm in a Sorvall SM-24 rotor. Interferon activity
in the supernatants was subjected to test for antiviral
activity.
Further purification may be effected as in the aforementioned
applications of Goeddel and Pestka.
The yields per liter of culture of these interferons, titrated on a
human cell line (WISH) are shown in Table 2 from which it is
apparent that LeIF-A/D activity is produced in greater amount than
the other interferons. This difference could be due to greater
intrinsic activity of the LeIF-A/D or to greater yield in terms of
mg protein of this interferon. Because the genetic link-up was
identical for all these interferons it seems most probable that
LeIF-A/D essentially has greater activity than the other
interferons.
TABLE 2 ______________________________________ YIELD OF LEUKOCYTE
INTERFERONS FROM SHAKING FLASK CULTURES OF E. COLI Type interferon
Activity Yield/Liter (Units on WISH)*
______________________________________ A 8 .times. 10.sup.7 D 5
.times. 10.sup.6 AD (Bgl II) 2 .times. 10.sup.8 DA (Bgl II) 1
.times. 10.sup.6 ______________________________________ *assayed by
inhibition of cytopathic effect on WISH cells with VSV as
challenge.
The potency of the various interferons in a range of mammalian cell
lines was determined (human, WISH; African green monkey, VERO;
hamster fibroblast, BHK; rabbit kidney cells, RK-13; mouse L-929;
and bovine kidney, MDBK cells). In order to compare the relative
activity of the interferons their activity on various cells was
calculated relative to their activity on WISH cells taken as 100.
The results in Table 3 show that LeIF-A/D has very high activity in
VERO and L-929 cells whereas LeIF-D/A has low activity in these
cell lines. These results indicate that the combination of the
N-terminal portion of LeIF-A and the C-terminal portion of LeIF-D
within one molecule (LeIF-A/D) confers to the hybrid protein
particular potency which is manifest in several mammalian species.
Moreover, these properties are not simply the summation of the
properties of the parent interferons. This is clearly seen in the
case of activity on L-929 cells (Table 3) in which case neither a
mixture of LeIF-A and LeIF-D nor the other hybrid, LeIF-D/A, has
significant activity.
TABLE 3 ______________________________________ TITRATION OF VARIOUS
LEUKOCYTE INTERFERONS IN CELL LINES FROM VARIOUS MAMMALIAN SPECIES
Leukocyte interferons* Cell line A D A/D D/A A + D Buffy-coat
______________________________________ WISH 100 100 100 100 100 100
VERO 250 75 1,670 20 200 200 BHK 400 200 833 2,000 400 20 RK-13 12
500 6 N.D. N.D. 120 L-929 150 5 3,300 2 10 0.1
______________________________________ *Interferons tested against
VSV infection of the different cell lines. Activities expressed as
percentage of activity observed in WISH cells.
The activity of LeIF-A/D against other viruses was also examined.
The data in FIG. 5 show antiviral effects against EMC virus
infection of L-cells and the data in FIG. 6 shows effects against
VSV infection of L-cells. It is clear from these data that the
greater activity of LeIF-A/D is not confined to one virus (VSV) and
its greater activity is likely to be a general property against
many viruses. Natural human buffy-coat interferon preparations have
no effect against mouse cells (see Table 2). The activity of
LeIF-A/D against EMC virus infection of CD-1 mice was therefore
examined. The results in FIG. 7 show that LeIF-A/D is extremely
potent against lethal EMC virus infection and LeIF-A also has
antiviral activity, as is to be expected from the activity in cell
lines (Table 2). The data in FIG. 7 result from treatments i.p. 3
hrs. before infection. Dosages of LeIF-A/D and LeIF-A are as
titrated on WISH.
Lethal EMC virus infection of hamsters is also affected by LeIF-A/D
and LeIF-A (FIG. 8), the former being the most effective, and buffy
coat interferon shows only a small and statistically insignificant
effect. In the case of FIG. 8, all interferons were given i.p. 3
hrs. before infection at a dose of 5.times.10.sup.5 .mu./kg,
titrated on WISH cells.
These results indicate that the pronounced antiviral effects of
LeIF-A/D in a range of mammalian species is not confined to cell
cultures but is also observed in lethal virus infections.
EMC virus can be considered a model system, a demonstration of
antiviral effect against which may be predictive of antiviral
effect against the represented family of viruses, e.g., the
picornavirus family of which foot and mouth disease and polio are
members. VSV virus can be considered a model system, a
demonstration of antiviral effect against which may be predictive
of antiviral effect against the represented family of viruses,
e.g., the rhabdovirus family of which rabies is the most important
member.
Table 4 tabulates the activities of various of the LeIF hybrids
hereof on WISH and MDBK cells and the activity ratios thereof:
TABLE 4 ______________________________________ Units/liter
Units/liter LeIF Hybrid culture culture Ratio Activities (PvuII)
WISH Cells MDBK Cells WISH/MDBK
______________________________________ AB 2.4 .times. 10.sup.8 4
.times. 10.sup.7 6 AD 1.2 .times. 10.sup.8 2 .times. 10.sup.7 6 AF
6 .times. 10.sup.7 1 .times. 10.sup.7 6 AG 4 .times. 10.sup.7 1.5
.times. 10.sup.7 2.7 BA 1.5 .times. 10.sup.7 1 .times. 10.sup.7 1.5
BD 6 .times. 10.sup.7 1.5 .times. 10.sup.7 4 BF 1 .times. 10.sup.6
3.5 .times. 10.sup.5 0.3 BG 2 .times. 10.sup.7 6 .times. 10.sup.7
0.3 DA 3 .times. 10.sup.6 1.2 .times. 10.sup.8 0.025 DB 2 .times.
10.sup.6 5 .times. 10.sup.7 0.04 DF 2 .times. 10.sup.5 4 .times.
10.sup.6 0.05 DG 2 .times. 10.sup.5 1.5 .times. 10.sup.7 0.014 FA 2
.times. 10.sup.5 6 .times. 10.sup.7 0.003 FB 2 .times. 10.sup.6 8
.times. 10.sup.7 0.025 FD 1 .times. 10.sup.7 2 .times. 10.sup.7 0.5
FG 1 .times. 10.sup.6 4 .times. 10.sup.7 0.025 A* 8 .times.
10.sup.7 1.2 .times. 10.sup.8 0.7 B* 8 .times. 10.sup.7 4 .times.
10.sup.8 0.2 C* 2 .times. 10.sup.7 1.5 .times. 10.sup.7 1.3 D* 5
.times. 10.sup.6 2.5 .times. 10.sup.7 0.2 F* 2 .times. 10.sup.7 2
.times. 10.sup.8 0.1 ______________________________________ *For
comparison purposes
APPENDIX A
THE STRUCTURE OF EIGHT DISTINCT CLONED HUMAN LEUKOCYTE INTERFERON
cDNAs
David V. Goeddel, David W. Leung, Thomas J. Dull, Mitchell Gross,
Richard M. Lawn, Russell McCandliss*, Peter H. Seeburg, Axel
Ullrich, Elizabeth Yelverton, and Patrick W. Gray
Department of Molecular Biology, Genentech, Inc., 460 Pt. San Bruno
Blvd., South San Francisco, California 94080
The interferons are a family of proteins which confer viral
resistance in target cells (1,2) as well as inhibiting cell
proliferation and modulating immune response (see refs. 2, 3 for
reviews). Human interferons were originally classified by their
respective cells of origin. The major interferons produced in
leukocytes (LeIF or IFN-.alpha.) and in fibroblasts (FIF or
IFN-.beta.) have been shown to be antigenically distinct,
acid-stable molecules collectively designated "classical" or type I
interferons. Intense clinical interest in interferon coupled with
the inability to obtain large amounts of the purified protein from
human cells has spurred efforts to produce high yields of human
interferon in the bacterium E. coli via recombinant DNA procedures.
Several groups have recently reported the bacterial synthesis of
various interferons (4-9) and, in one instance, the efficacy of
such material in limited animal testing has been demonstrated
(5).
We recently described the construction of a collection of cDNA
clones prepared using 12S RNA isolated from a human myeloblastoid
cell line (5). Several cDNA clones were identified by hybridization
with synthetic DNA probes designed from partial amino acid sequence
information of human leukocyte interferon. The cDNA insert of one
of the hybrid plasmids (pL31) was sequenced and used to construct a
plasmid which directed the synthesis of large amounts of a
biologically active human LeIF, termed LeIF A (5). Restriction
endonuclease analysis showed extensive differences between the
additional hybridizing clones, suggesting that several different
human LeIFs might be expressed by the myeloblastoid cell line KG-1
(10). Furthermore, pL31 differs considerably from the human LeIF
cDNA clone described by Weissmann and colleagues (4) in both
nucleotide sequence and deduced amino acid sequence; yet both of
the encoded LeIFs display antiviral activity (4,5).
Additional evidence for the existence of a multigene family of
human LeIFs is found in the heterogeneity of the IF proteins
prepared from human leukocytes (11) and lymphoblasts (12,13).
NH.sub.2 -terminal amino acid sequences (12-14) and amino acid
sequences of tryptic and chymotryptic peptides (13) have revealed
at least five distinct, but homologous, LeIFs. At least eight
distinct chromosomal LeIF genes have recently been isolated from a
human gene library (15; R.L. and A.U., unpublished results). In
contrast, there is at present no evidence for multiple human FIF
genes. NH.sub.2 -terminal amino acid sequences of FIF determined by
two different groups (16,17) are in agreement with results obtained
from nucleotide sequencing of cloned FIF cDNAs (7,18,19) and FIF
mRNA (20,21) in suggesting that there may be only one gene for FIF.
We describe here genomic hybridization data consistent with the
existence of approximately ten human LeIF genes and a single FIF
gene, and present the DNA sequences of eight distinct LeIF cDNA
clones identified in a cDNA library prepared from the myeloblastoid
cell line KG-1.
Evidence for Multiple LeIF Genes
To estimate the number of LeIF genes in the human genome, human
spleen DNA was digested with various restriction endonucleases,
electrophoresed through agarose gels and transferred to
nitrocellulose paper (22). Hybridizations were performed with
radiolabelled LeIF and FIF (7) cloned cDNAs.
Identification and Classification of Multiple LeIF cDNA Clones
To investigate the multiplicity of cloned human LeIF cDNAs an in
situ colony screening procedure (23) was performed on 4,000
individual colonies of our cDNA library. Approximately 50 clones
hybridized to a 260 base pair BglII fragment from pL31 encoding
amino acids 61 through 150 of LeIF A. 33 of the 50 contained cDNA
inserts of 700 base pairs or longer and were selected for further
analysis. The cDNA inserts were mapped using the restriction
enzymes PvuII, BglII, and EcoRI, since PvuII and BglII had been
found to cleave LeIF A cDNA (5) and all three cut the LeIF cDNA
cloned by Nagata et al. (4). The restriction endonuclease maps thus
obtained permitted us to divide the LeIF cDNAs into 8 groups The
largest cDNA inserts in each group were sequenced by a combination
of the Maxam-Gilbert chemical procedure (24) and the dideoxy chain
termination procedure using the bacteriophage M13 cloning vector
mp7 (25). It remained possible that different LeIF cDNAs would
display the same restriction enzyme map. Since the 3'-noncoding
regions of the 8 sequenced cDNA clones showed much less homology
than their respective coding portions, the 3' regions of several
cDNAs in each group were partially sequenced. This analysis
revealed that one of the cDNA clones originally classified as type
C was actually a LeIF H cDNA clone which did not extend to the
single 5' PvuII site. Fourteen of the 33 LeIF cDNA clones analyzed
were LeIF A and 9 were LeIF D. No other type of clone was
represented more than twice in this collection. This result was not
an artifact of the use of LeIF A cDNA as the original hybridization
probe. No additional LeIF cDNA clones were found when the 4,000
colonies of the cDNA library were re-tested using mixed LeIF A, B,
C, E and F cDNA probes.
LeIF D has the same restriction pattern as the LeIF cDNA clone
described by Nagata et al. (4). The only difference between the two
clones in predicted amino acid sequence is at position 114 where
valine is found in LeIF D instead of an alanine (26). Weissmann and
coworkers (6) have also recently sequenced a partial length LeIF
cDNA clone of a second type (which they named .alpha..sub.2)
differing from LeIF A only at the codon for amino acid 23 (arginine
for .alpha..sub.2 vs. lysine for LeIF A).
A LeIF Pseudogene is Transcribed
The coding region of the LeIF E cDNA clone contains one nucleotide
more than the other LeIF cDNAs. The predicted amino acid sequence
from this nucleotide insertion onwards (amino acid residue 40 in
LeIF E) bears no resemblance to the other LeIF protein sequences
and results in several in-phase termination codons, the first of
which would appear at amino acid position 60. Therefore, the amino
acid sequence is presented in FIG. 4 as if this insertion had not
occurred. Even with this adjustment, termination codons are
encountered at positions 102 and 155. Hence, termination codons
occur in all three reading frames. The fact that a cDNA was cloned
which contains an additional nucleotide in the coding sequence for
a LeIF may reflect an artifact created somewhere during the process
of reverse transcription of mRNA and sequence amplification in E.
coli. However, the presence of the two additional termination
codons could imply that LeIF E is a copy of a pseudogene analagous
to the .alpha.- and .beta.-globin pseudogenes (27-29). If so, this
is the first reported evidence of a pseudogene being transcribed
into RNA. The biological significance of the transcription and
possible translation of this pseudogene has not been determined.
The fact that only one cDNA clone of a LeIF pseudogene was obtained
in our extensive search for LeIF cDNA clones suggests that the
transcription of such genes may be very rare. This observation is
further supported by our recent isolation from a human chromosomal
DNA library of an additional LeIF pseudogene having a DNA sequence
distinct from the eight LeIF cDNA clones described here
(unpublished results).
LeIF Signal Peptides
The signal sequences of human LeIFs A, B, C, D, F and H possess
features common to all eukaryotic secretory protein signal
sequences. Each contains a long stretch of hydrophobic amino acids
residues, perhaps important for association with and traversal of
the rough endoplasmic reticulum membrane. The hydrophobic regions
are flanked by charged and more polar amino acid residues. The
signal sequences are rich in serine residues proximal to the
prepeptide cleavage site, analogous to the signal sequences of rat
proalbumin (30) and certain mouse immunoglobin chains (31). Within
the cell, removal of the signal peptides is presumably achieved by
peptide cleavage between glycine and cysteine residues in all of
these LeIFs.
The signal sequences of many different proteins have been
determined and compared (32). No major sequence homologies have
been detected; the most general feature may be structural in nature
(see ref. 33 for review). It is possible that the junction of
distinct structural domains within the signal peptide is recognized
by a processing enzyme or signal peptidase (34), resulting in
production of the mature secreted protein and complete
translocation into the lumen of the rough endoplasmic
reticulum.
The signal sequences of the LeIFs reported here are only about 70
percent homologous when compared with each other, except for types
C and F which are identical (Table 1). Only 11 of the 23 amino
acids (43 percent) occur in invariant positions in all six
prepeptides. The sequence of the human fibroblast interferon signal
peptide (7,18-20) is completely different from those of the human
LeIFs.
Mature Leukocyte Interferons
The mature LeIFs are more closely related in their amino acid
sequences (approximately 80 percent homology) than are their
corresponding signal peptides (See Table 1). A total of 79 out of
166 amino acids (48 percent) of the mature polypeptide are
identical in all sequences examined. If the LeIF E pseudogene amino
acid sequence is ignored then 99 amino acids (60 percent) occupy
identical positions. When the human FIF sequence (7,18,19,21) is
compared with the LeIF sequences, only 39 amino acids out of 166
(23 percent) appear in identical positions. The overall
distribution of amino acid differences along the LeIF molecules
appears to be random with the possible exception of the region
between amino acids 115 and 151. All the LeIF cDNA clones (again
excluding LeIF E) code for the same amino acid in 31 out of 37 (84
percent) positions in this region. Residues 116 to 150 also display
the greatest similarity to FIF (17 out of 35 positions identical),
suggesting that this region is of functional importance.
The changes in amino acid sequences between the LeIFs are largely
due to single nucleotide changes; there are several sites (i.e.
positions 37, 107, 160) where nonconservative amino acid changes
occur. It is interesting to note that the nucleotide sequence of
LeIF B contains an insertion of a thymidine residue after
nucleotide 362 (codon 98). This insertion changes the translational
reading frame and results in four distinct codon changes. Then
after position 373 (codon 101) an adenosine residue has been
deleted which restores the original reading frame:
______________________________________ 97 102
______________________________________ LeIF A glu ala cys val ile
gln GAA GCC TGT GTG ATA CAG LeIF B glu val leu cys asp gln GAA GTC
CTG TGT GAT CAG ______________________________________
The shorter (.about.800 base pairs) of the two LeIF H type cDNA
clones, which we have designated LeIF H.sub.1, has a deletion
(cytidine, nucleotide number 545) which is followed 6 nucleotides
later by a correcting insertion (guanosine, after position
551):
______________________________________ 158 162
______________________________________ LeIF H leu gln lys arg leu
TTG CAA AAA AGA TTA LeIF H.sub.1 leu lys lys gly leu TTG AAA AAA
GGA TTA ______________________________________
Except for this difference and a single base change in the 3'
noncoding regions, the nucleotide sequences of LeIF H and LeIF
H.sub.1 cDNAs are identical. It is possible these two cDNA clones
may represent allelic forms of a single LeIF gene found in the KG-1
cell line.
The location of cysteine residues is often highly conserved in
proteins. All the LeIFs which we have sequenced code for cysteines
at positions 1 and 139 of the mature protein. All predicted LeIFs
except LeIF E contain a cysteine at position 29; all except LeIF B
(Cys-100) contain a cysteine at position 99. At least one
intramolecular disulfide bridge has been predicted on the basis of
loss of interferon activity upon incubation with reducing agents
(35). Allen and Fantes (13) have shown that the N-terminal cysteine
is probably involved in a disulfide bond. Furthermore, the presence
of two disulfide bridges in the LeIF A produced in E. coli (5) has
been established by analysis of tryptic digests of the purified
protein: Cys-1 is bonded to Cys-99 and Cys-29 is bonded to Cys-139
(R. Wetzel, personal communication). The fact that cysteine
residues are also predicted in human FIF (7,18,19,21) in the
positions corresponding to amino acids 29 and 139 of LeIF suggests
that this disulfide bond also occurs in FIF.
NH.sub.2 -terminal sequences of human LeIF species isolated from
lymphoblasts (12,13) and leukocytes (14) have been determined by
protein microsequence analysis. The sequences determined by Zoon et
al. (12) and Levy et al. (14) both have NH.sub.2 -terminal serine
residues. The sequences published by Allen and Fantes (13) begin
with cysteine residues. The seven full length LeIF cDNAs (A-F, H)
all code for mature LeIFs beginning with cysteine. While it is
possible that a LeIF cDNA clone will be identified which encodes an
N-terminal serine, it is more likely that the determination of
serine arose by artifactual oxidation of cysteine during the
initial protein microsequencing procedure.
Disregarding the discrepancies at amino acid position one, the
following comparisons can be made. The 32 NH.sub.2 -terminal amino
acids of the lymphoblastoid IF sequence of Zoon et al. (12) have
the greatest homology with LeIF F, with only position 26 (Pro in
LeIF F, Leu in lymphoblastoid IF) differing between the two
species. The 22 NH.sub.2 -terminal amino acids of LeIF determined
by Levy et al. (14) show the most homology with LeIF A. These two
LeIFs differ only at position 11 (Asn in the Levy sequence vs. Ser
for LeIF A). Allen and Fantes have determined the sequences of
tryptic and chymotryptic peptides of two forms of LeIF,
IFN-.alpha.A and IFN-.alpha.B (13). IFN-.alpha.A consists of at
least two different polypeptides of 165 amino acids having the same
amino acid deletion at position 44 that is predicted by the LeIF A
cDNA sequence. 140 of the 142 amino acids which have been
determined from the IFN-.alpha.A peptides correspond to the LeIF A
cDNA sequence presented here. Only Asp-84 and Pro-88 of
IFN-.alpha.A are not predicted by the LeIF A cDNA sequence.
IFN-.alpha.B consists of at least three polypeptides of 166 amino
acids each and the residues present at 161 positions have been
determined (13). Peptides of IFN-.alpha.B are most closely related
to LeIF D, with only Met-60, Thr-64, and Ala-114 not being
predicted by the LeIF D cDNA sequence. The 77 N-terminal amino
acids of LeIF F are found in the IFN-.alpha.B peptide sequences.
However, there are eleven differences between these two interferon
sequences in the C-terminal portion (residues 78-166) of the
polypeptides. Amino acid sequences deduced from LeIF B, C, G and H
cDNAs do not appear to be represented in either IFN-.alpha.A or
IFN-.alpha.B peptide sequences. These comparisons seem to suggest
the presence of additional LeIF genes, cDNA copies of which are not
present, or have not been identified, in our cDNA library. Whether
some of the slight differences among LeIF sequences described above
could be due to allelic variation in the human population awaits a
detailed genomic characterization of the LeIF gene family from an
individual.
Noncoding Sequences of LeIF
A limited amount of sequence information is available for the 5'
noncoding regions of the LeIF cDNAs. Obvious homologies exist in
the regions immediately preceding the translational initiation
codon; LeIFs A, B and D are similar in this region, as are LeIFs C
and F.
The 3' noncoding regions of the LeIF cDNA clones vary in size from
242 (LeIF D) to 441 (LeIF B) base pairs, counting from the first
nucleotide of the stop codon to the site of polyadenylation. The
overall homology between 3' noncoding regions is lower than that
within the protein coding regions, although certain portions of the
3' noncoding region are relatively conserved. Both the reduced
level of 3' noncoding homology and the substantial deletions in the
sequence alignments resemble the situation found in comparing the
analogous DNA sequences of members of the human .beta.-like globin
gene family (36). As those authors point out, such findings suggest
that the precise sequence of much of the 3' noncoding regions of
eukaryotic mRNAs is not essential for function. Experiments
involving in vitro translation of mRNAs with large 3' deletions
support this contention (37,38).
Efstratiadis and coworkers (36) have noted that short (2-8 bp)
tandem repeats often surround the sites of deletions in the
.beta.-like globin gene family 3' noncoding sequences. They
suggested a mechanism of "slippage" during DNA replication followed
by scission of resulting single stranded DNA loops as a possible
cause of these deletion events during evolution. Such short tandem
repeats also occur at many of the deletion points in the LeIF cDNA
sequences.
The hexanucleotide AATAAA precedes the site of polyadenylation in
many eukaryotic cellular mRNAs by 15-25 bases (39). Although
exceptions to the rule have been found in several RNA viruses (40),
this sequence is thought to be involved in mRNA processing and/or
polyadenylation. A variant of this sequence, AATTAAA, has recently
been reported which proceeds the poly(A) site in rat amalyase (41)
and anglerfish somatostatin (42) mRNAs. LeIF A, D and F cDNAs
contain the hexanucleotide AATAAA ending 20, 21 and 14 nucleotides,
respectively, from their polyadenylation sites. LeIF B, C, E, G and
H cDNAs do not contain this hexanucleotide. At the nucleotide
position corresponding to AATAAA in the two shortest mRNAs (A and
D), the sequence of the other mRNAs differ by one or two
nucleotides. This sequence divergence may have led to the extension
of the 3' non-coding region in those genes. While LeIF F mRNA
continues until an AATAAA is encountered 14 nucleotides before its
poly(A) site, LeIF C, E, G and H possess the related hexanucleotide
ATTAAA near their 3' termini (positions 378-383). LeIF B also has
the ATTAAA sequence at this position, but is not polyadenylated
until another ATTAAA sequence is reached at positions 463-468. It
should be noted that the two cDNA clones most often isolated (LeIF
A and D) possess the canonical hexanucleotide at the same position
and cDNA clones corresponding to the other LeIFs are rare.
Therefore, it is conceivable that transcription of the genes for
the rarer mRNA species might often continue until a stronger
polyadenylation signal is reached, resulting in LeIF mRNA species
larger than 12S. However, none of the 10 total rare LeIF cDNA
clones (types B, C, E, F and H) we have isolated contain additional
3' noncoding nucleotides.
Significance of the LeIF multigene family
We have presented here the sequences of eight distinct human LeIF
cDNAs, seven of which probably code for functional interferon
polypeptides. LeIF amino acid sequence information (12-14) suggests
the presence of yet additional LeIF structural genes. Genomic blots
and the isolation of chromosomal LeIF sequences (15) also support
the concept of a LeIF multigene family consisting of at least 8-10
distinct human LeIF-related genes, although some of these could be
pseudogenes similar to LeIF E.
The cDNA clones LeIF A and LeIF D occur relatively frequently in
our cDNA library (5) which was prepared using RNA from the
virus-induced myeloblastoid cell line KG-1 (10). In contrast, a
major LeIF species found in the lymphoblastoid Namalwa cell line
(12) most closely resembles the polypeptide encoded by LeIF F cDNA.
This finding supports the suggestion of Nagata et al. (15) that
different cell types might preferentially express different LeIF
genes. Preferential expression of different interferons might also
occur in the same cell line in response to different viruses.
These multiple LeIFs probably exhibit distinct functional
properties. Streuli et al. (6) have shown that two different LeIFs
(IFN-.alpha.1 and IFN-.alpha.2) have striking differences in their
antiviral activities on cells of different species. We have
recently obtained expression in E. coli of the mature LeIF
polypeptides encoded by LeIF A, B, C, D, and F cDNAs (unpublished
results). All five of these interferons exhibit different
cross-species specificities. The availability of these cloned LeIFs
should permit the assignment of the various activities
characteristic of mixed leukocyte interferon preparations (2) to
individual LeIF species. A detailed understanding of the
correlation between LeIF structure and activity may lead to the
design of new interferons highly active against particular
diseases.
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TABLE 1 ______________________________________ Pairwise comparisons
of differences in coding sequences of human LeIFs
______________________________________ ##STR1##
______________________________________ *Only the 133 Cterminal
amino acids of LeIF G can be deduced from the cDN sequence.
Therefore, in pairwise comparisons involving LeIF G only
differences in these 133 amino acids are considered.
The number of amino acid replacements in each pair of coding
sequences are shown. The 23 amino acid signal peptides are compared
in the lower left and the 166 amino acid mature LeIFs are compared
in the upper right of the Table. The table lists first the total
number of differences between pairs, followed by the percentage
difference (numbers in parentheses). Signal sequences for LeIF E
and LeIF G are not known, so no entries appear for comparisons
involving either of these sequences.
* * * * *