Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1999-11-08
2002-06-25
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Vector, per se
C435S425000, C536S023100
Reexamination Certificate
active
06410315
ABSTRACT:
BACKGROUND OF THE INVENTION
The invention relates to methods for identifying genes encoding signal sequences.
The demonstrated clinical utility of certain growth factors and cytokines, for example, insulin, erythropoietin, granulocyte-colony stimulating factor, granulocyte-macrophage colony stimulating factor, human growth hormone, interferon-beta, and interleukin-2 in the treatment of human disease has generated considerable interest in identifying novel proteins of this class.
Since growth factors and cytokines are secreted proteins, they often possess “signal sequences” at their amino terminal end. The signal sequence directs a secreted or membrane protein to a sub-cellular membrane compartment, the endoplasmic reticulum, from which the protein is dispatched for secretion from the cell or presentation on the cell surface. Techniques that detect signal sequences or nucleic acid sequences encoding a signal sequence have been employed as tools in the discovery of novel cytokines and growth factors.
Among the methods that have been used to identify secreted proteins are methods that rely on the homology between some secreted proteins. For example, DNA probes or PCR oligonucleotides that recognize sequence motifs present in genes encoding known secreted proteins have been used in screening assays to identify novel secreted proteins. In a related approach, homology-directed sequence searching of Expressed Sequence Tag (EST) sequences generated by high-throughput sequencing of specific cDNA libraries has been used to identify genes encoding secreted proteins. Both of these approaches can identify a signal sequence when there is a high degree of similarity between the DNA sequence used as a probe and the putative signal sequence.
“Signal peptide trapping” has also been used to identify secreted proteins (Tashiro et al., 1993, Science 261:600-603; Honjo et al., 1996; U.S. Pat. No. 5,525,486, and U.S. Pat. No. 5,536,637). Generically, this technique involves the ligation of cDNA, prepared from various mRNA sources, to a reporter gene lacking a signal sequence. The resulting chimeric constructs are introduced into an appropriate host cell. Depending upon the nature of the reporter gene, host cells are scored for either the presence of reporter protein at the cell surface or secretion of the reporter protein from cells. In both cases, a positive score indicates that the cell harbors a chimeric construct having a cDNA encoding a signal sequence which directs the export of the reporter protein to the cell surface or into the extracellular medium.
In a related method (Klein et al., 1996, Proc. Nat. Acad. Sci. USA 93:7108-7113; Jacobs, 1996, U.S. Pat. No. 5,536,637) the Saccharomyces cerevisiae gene, SUC2, which encodes a secreted invertase protein, is used as a reporter. Invertase catalyzes the hydrolysis of sucrose into glucose and fructose, sugars which, unlike sucrose, can be readily utilized by
S. cerevisiae
as a carbon source. Strains of
S. cerevisiae
that cannot secrete SUC2 protein are unable to grow on media with sucrose as the sole carbon source. Thus, a mutant SUC2 gene which does not encode a signal peptide can be used as a reporter in signal sequence trapping. Chimeric constructs composed of random cDNAs fused to DNA encoding SUC2 lacking a signal sequence are transformed into
S. cerevisiae
, and transformants secreting chimeric SUC2 are selected by growing the transformants under conditions where sucrose is the sole carbon source. This method offers a genetic selection for cDNAs encoding signal peptides.
SUMMARY OF THE INVENTION
The invention features a method for identifying nucleic acid sequences encoding signal sequences. Most secreted and membrane-associated proteins possess such signal sequences composed of 15-30 hydrophobic amino acid residues at their amino termini. Because signal sequences are present in secreted proteins and membrane-associated proteins, the identified nucleic acid sequences, which will encode at least a portion of a secreted or membrane-associated protein, can be used to isolate additional nucleic acid molecules encoding the entirety of the secreted or membrane-associated protein.
KRE9 is an example of a yeast secreted protein. Yeast KRE9 null mutants show severe growth retardation (essentially no growth) when glucose is the sole carbon source. Growth of a KRE9 null mutant on glucose can be restored by transformation with DNA encoding wild type KRE9 protein, but not by transformation with DNA encoding a mutant KRE9 protein lacking a signal sequence. Thus, secretion of KRE9 protein via its signal sequence is required for its normal function. Importantly, the presence of extracellular KRE9 protein does not rescue the KRE9 null phenotype. This result suggests that KRE9 protein must pass through the secretory pathway in order to exert its normal function. Although yeast KRE9 null mutants show essentially no growth when glucose is used as the carbon source, they can be maintained on galactose because of induction of the KNH1, a functional homolog of KRE9.
The invention features a method for identifying secreted and membrane-associated proteins using yeast that lack functional KRE9 protein and are transformed with a chimeric DNA molecule in which a mutant KRE9 gene lacking its signal sequence encoding portion is fused to a test sequence. The transformed yeast are grown on a selective medium that is designed permit (or prevent) growth of cells which produce functional, secreted KRE9. If the test sequence encodes a signal sequence (fused in-frame to the sequence encoding mature KRE9 protein), the yeast cell will grow (or not grow in the case of a selective medium which is designed to prevent growth of cells expressing functional, secreted KRE9) on the selective medium. Thus, the invention features a novel selection method utilizing DNA constructs containing a chimeric KRE9 gene in which the part of the KRE9 gene encoding the native KRE9 signal sequence is replaced with a candidate signal sequence encoding sequence. The ability of these chimeric constructs to rescue KRE9 null mutants grown on glucose is tested as follows. The chimeric constructs are used to transform KRE9 null mutants. The transformed cells are transferred to plates having glucose as the sole carbon source. Those chimeric constructs that allow a transformed KRE9 null mutant to grow on glucose contain candidate signal sequence encoding sequences.
Since growth factors and cytokines are secreted proteins, possessing signal sequences at their amino termini, signal sequence trapping can be employed as a tool in the discovery of novel proteins of this class.
One embodiment of the methods of the invention includes the following steps:
(a) obtaining a nucleic acid molecule which includes a chimeric gene, the chimeric gene including a first portion and a second portion, the first portion encoding a KRE9 lacking a functional signal sequence and the second portion being a heterologous nucleic acid sequence;
(b) transforming a yeast cell lacking a functional KRE9 gene with the nucleic acid molecule; and
(c) determining whether the transformed yeast cell grows when supplied with a medium that permits growth of a yeast cell expressing KRE9 having a functional signal sequence, but does not permit growth of a yeast cell that does not express KRE9 having a functional signal sequence, wherein growth on the medium indicates that the heterologous nucleic acid sequence present in the yeast cell encodes a signal sequence.
In another embodiment the method, step (a) includes:
(i) obtaining double-stranded DNA; and
(ii) ligating the double-stranded DNA to a DNA molecule encoding KRE9 lacking a functional signal sequence to create a chimeric gene.
In another embodiment of the invention step (a) includes:
(i) obtaining double-stranded DNA;
(ii) ligating the double-stranded DNA to a DNA molecule encoding KRE9 lacking a functional signal sequence to create a chimeric gene;
(iii) transforming a bacterium with a nucleic acid molecule that includes the chimeric gene;
(iv) growing the transformed bacterium; and
(v) is
Bossone Steven
McCarthy Sean A.
McKelvey Terry
Millennium Pharmaceuticals Inc.
Millennium Pharmaceuticals Inc.
Sandals William
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