We claim:
1. An electrical connector assembly comprising:
(a) a hollow bored adapter having a forward end and a rearward end, the forward end comprising electrical component attaching means, the rearward end comprising a hollow bored nipple, the hollow bore of the nipple being continuous with the hollow bore of the adapter, the hollow bored nipple having an outwardly opening channel;
(b) at least a first electrical cable extending through the bore of the hollow bored adapter, and extending through the bore of the hollow bored nipple, the at least first electric cable extending rearwardly from the rearward end of the hollow bored adapter;
(c) a flexible tubular sheath having a forward end overlying the hollow bored nipple’s outwardly opening channel, the flexible tubular sheath extending rearwardly from the rearward end of the hollow bored adapter; and,
(d) a thermoplastic strap capable of alternately assuming a longitudinally stretched configuration, and a shortened plastic memory configuration, the thermoplastic strap being spirally wrapped about the flexible tubular sheath and overlying the hollow bored nipple’s outwardly opening channel.
2. The electrical connector assembly of claim 1 wherein the thermoplastic strap is in its plastic memory configuration and annularly nests within the outwardly opening channel.
3. The electrical connector assembly of claim 2 wherein the thermoplastic strap has inner and outer ends, the outer end of the polymer strap being adhesively secured to an immediately underlying wrap of the thermoplastic strap.
4. The electrical connector assembly of claim 3 wherein the thermoplastic strap comprises a material selected from the group consisting of polyester and polyimide.
5. The electrical connector assembly of claim 1 wherein the hollow bored nipple has a non-circular cross-sectional shape.
6. The electrical connector assembly of claim 5 wherein the outwardly opening channel has a rearward end defined by a first outwardly extending ridge having a knurled or ridged outer surface.
7. The electrical connector assembly of claim 6 wherein the outwardly opening channel has a knurled or ridged floor.
8. The electrical connector assembly of claim 7 wherein the outwardly opening channel has a forward end defined by a second outwardly-extending ridge.
9. The electrical connector assembly of claim 1 wherein the electrical component attaching means comprises a connector selected from the group consisting of mounting flanges, rotatable coupling nuts, helical threads, mounting lugs, mounting lug receiving slots, and mounting clips.
10. The electrical connector assembly of claim 9 wherein the flexible tubular sheath comprises a material selected from the group consisting of metal wire braid, polymer fiber braid, glass fiber braid, and polymer tubing.
The claims below are in addition to those above.
All refrences to claim(s) which appear below refer to the numbering after this setence.
We claim:
1. An isolated andor recombinant SIP ligase.
2. An isolated andor recombinant cdc4 polypeptide, which polypeptide comprises an amino acid sequence identical or homologous to the amino acid sequence designated by SEQ ID No. 2 or 4.
3. The cdc4 polypeptide of claim 2, which polypeptide has the amino acid sequence encoded by a nucleic acid which hybridizes under stringent conditions to the nucleotide sequence set forth in SEQ ID NO. 1.
4. The cdc4 polypeptide of claim 2, which polypeptide has an endogenous ubiquitin ligase activity andor is a component of a ubiquitin ligase.
5. The cdc4 polypeptide of claim 2, wherein the cdc4 polypeptide mediates ubiquitination of a cell-cycle regulatory protein.
6. The cdc4 polypeptide of claim 5, wherein the cell-cycle regulatory protein is a CDK inhibitor.
7. The cdc4 polypeptide of claim 6, wherein the cyclin-dependent kinase inhibitor is p27kip1.
8. The cdc4 polypeptide of claim 5, wherein the cell-cycle regulatory protein is a cyclin.
9. The cdc4 polypeptide of claim 1, which polypeptide is capable of interacting with at least one other protein selected from the group consisting of a component of a ubiquitin ligase, a skp1 protein, a ubiquitin conjugating enzyme, and a p27 protein.
10. The cdc4 polypeptide of claim 1, which polypeptide is at least 75% homologous to the amino acid sequence designated by SEQ ID NO. 2.
11. The cdc4 polypeptide of claim 1, which polypeptide is a vertebrate cdc4 protein.
12. The cdc4 polypeptide of claim 11, which polypeptide is a mammalian cdc4 protein.
23. An isolated nucleic acid which selectively hybridizes under high stringency conditions to at least ten nucleotides of a nucleic acid sequence represented by one of SEQ ID NO. 1 or SEQ ID NO. 3, or complementary sequences thereof, which nucleic acid can specifically detect or amplify a nucleic acid sequence of an vertebrate cdc4 gene.
24. The nucleic acid of claim 23, which is nucleic acid is labeled.
25. A reconstituted protein mixture comprising an a SIP polypeptide and a cell-cycle regulatory protein.
26. The protein mixture of claim 25, wherein the cell-cycle regulatory protein is a CDK inhibitor.
27. The cdc4 polypeptide of claim 26, wherein the cyclin-dependent kinase inhibitor is p27kip1.
28. The cdc4 polypeptide of claim 25, wherein the cell-cycle regulatory protein is a cyclin.
29. An isolated SIP polypeptide having a ubiquitin moiety attached to a cysteine thereof.
30. An assay for identifying an inhibitor of SIP-mediated ubiquitination, comprising:
(i) providing a ubiquitin-conjugating system including the substrate polypeptide, a SIP ligase and ubiquitin, under conditions which promote ubiquitination of the substrate polypeptide by the SIP ligase;
(ii) contacting the ubiquitin-conjugating system with a candidate agent;
(iii) measuring a level of ubiquitination of the substrate polypeptide in the presence of the candidate agent; and
(iv) comparing the measured level of ubiquitination in the presence of the candidate agent with ubiquitination of the substrate polypeptide in the absence of the candidate agent,
wherein a statistically significant decrease in ubiquitination of the substrate polypeptide in the presence of the candidate agent is indicative of an inhibitor of SIP-mediated ubiquitination.
31. The assay of claim 30, wherein the ubiquitin-conjugating system comprises a reconstituted protein mixture.
13. The cdc4 polypeptide of claim 12, which polypeptide has the amino acid sequence set forth in SEQ ID NO. 2.
14. An isolated nucleic acid comprising a nucleotide sequence encoding an cdc4 polypeptide, or a nucleotide sequence complementary thereto, said cdc4 polypeptide including an amino acid sequence identical or homologous to the amino acid sequence set forth in SEQ ID NO. 2 or SEQ ID NO. 4, or a portion thereof.
15. The nucleic acid of claim 14, which nucleic acid hybridizes under stringent conditions to a nucleic acid probe having a nucleotide sequence represented by at least 60 consecutive nucleotides of SEQ ID NO. 1 or 3, or a sequence complementary thereto.
16. The nucleic acid of claim 15, which nucleic acid comprises the nucleotide sequence set forth in SEQ ID NO. 1.
17. An isolated nucleic acid comprising a nucleotide sequence encoding an vertebrate SIP polypeptide.
18. The nucleic acid of claim 14 or 17, further comprising a transcriptional regulatory sequence operably linked to said nucleotide sequence so as to render said nucleic acid suitable for use as an expression vector.
19. An expression vector, capable of replicating in at least one of a prokaryotic cell and eukaryotic cell, comprising the expression vector of claim 18.
20. A host cell transfected with the expression vector of claim 18 and expressing said recombinant polypeptide.
21. A method of producing a recombinant cdc4 polypeptide comprising culturing the cell of claim 20 in a cell culture medium to express said recombinant polypeptide and isolating said recombinant polypeptide from said cell culture.
22. A transgenic animal having cells which harbor a transgene comprising the nucleic acid of claim 14 or 17, or in which a gene comprising said nucleic acid is disrupted.
32. The assay of claim 30, wherein the ubiquitin-conjugating system comprises a cell lysate.
33. The assay of claim 30, wherein the substrate polypeptide includes a ubiquitination sequence of a cell-cycle regulatory protein.
34. The assay of claim 33, wherein the cell-cycle regulatory protein is a CDK inhibitor.
35. The assay of claim 34, wherein the CDK inhibitor is a CIPKIP protein.
36. The assay of claim 35, wherein the CDK inhibitor is p27kip1.
37. The assay of claim 33, wherein the cell-cycle regulatory protein is a cyclin.
38. The assay of claim 30, wherein the SIP ligase is a cdc4 ligase.
39. The assay of claim 30, wherein the cdc4 ligase is a human cdc4 ligase.
40. The assay of claim 30, wherein the ubiquitin is provided in a form selected from a group consisting of:
(i) an unconjugated ubiquitin, in which case the ubiquitin-conjugating system further comprises an E1 ubiquitin-activating enzyme (E1), an E2 ubiquitin-conjugating enzyme (E2), and adenosine triphosphate;
(ii) an activated E1:ubiquitin complex, in which case the ubiquitin-conjugating system further comprises an E2;
(iii) an activated E2:ubiquitin complex; and
(iv) an activated ubiquitin complex with the SIP ligase.
41. The assay of claim 30, wherein the ubiquitin-conjugating system further comprises an E2 ubiquitin conjugating enzyme.
42. The assay of claim 30, wherein at least one of the ubiquitin and the substrate polypeptide comprises a detectable label, and the level of ubiquitination ot the substrate polypeptide is quantified by detecting the label in at least one of the substrate polypeptide, the ubiquitin, and ubiquitin-conjugated substrate polypeptide.
43. The assay of claim 42, wherein the label group is selected from a group consisting of radioisotopes, fluorescent compounds, enzymes, and enzyme co-factors.
44. The assay of claim 42, wherein the detectable label comprises a polypeptide having a measurable activity, and the substrate polypeptide is fusion protein including the detectable label.
45. The assay of claim 30, wherein the amount of ubiquitination of the substrate polypeptide is quantified by an immunoassay.
46. The assay of claim 30, wherein the amount of ubiquitination of the substrate polypeptide is quantified by an chromatography or electrophoresis.
47. The assay of claim 30, wherein the ubiquitin-conjugating system comprises a host cell expressing the substrate polypeptide and SIP ligase.
48. The assay of claim 47, wherein the host cell expresses a recombinant SIP ligase.
49. The assay of claim 30, wherein the ubiquitin-conjugating system comprises a cullins polypeptide.
50. An assay for identifying an inhibitor of an interaction between an substrate polypeptide and a SIP protein, comprising:
(i) providing a reaction system including the substrate polypeptide and a SIP protein, under wherein the substrate polypeptide and the SIP protein interact;
(ii) contacting the reaction system with a candidate agent;
(iii) measuring formation of complexes containing the substrate polypeptide and the SIP protein in the presence of the candidate agent; and
(iv) comparing the measured formation of complexes in the presence of the candidate agent with complexes formed in the absence of the candidate agent,
wherein a statistically significant decrease in the formation of complexes in the presence of the candidate agent is indicative of an inhibitor of the interaction of the substrate polypeptide and the SIP protein.
51. The assay of claim 50, wherein the reaction system comprises a reconstituted protein mixture.
52. The assay of claim 50, wherein the reaction system comprises a cell lysate.
53. The assay of claim 50, wherein the reaction system comprises a cell.
54. The assay of claim 50, wherein the substrate polypeptide and the SIP protein are provided as fusion proteins in an interaction trap system.
55. The assay of claim 50, wherein the substrate polypeptide is p27kip1.
56. The assay of claim 50, wherein the SIP protein is mutated to lack endogenous ubiquitination activity.
57. The assay of claim 30, which comprises a further step of preparing a pharmaceutical preparation of one or more compounds identified as inhibitors of the SIP-mediated ubiquitination.
58. A method for diagnosing a hyperproliferative disorder in a patient which disorder is associated with the destabilization of a CKI protein in cells of the patient, comprising: (i) ascertaining the level of expression of a SIP ligase andor the level of ligase activity of the SIP ligase in a sample of cells from the patient; and (ii) diagnosising the presence or absence of a hyperproliferative disorder utilizing, at least in part, the ascertained level expression or activity of the ligase, wherein an increase level of a SIP protein or SIP ligase activity in the sample, relative to a normal control sample of cells, correlates with the presence of a hyperproliferative disorder.
59. A prognostic method for evaluating the aggressiveness andor rate of recurrence of a disorder marked by aberrant hyperproliferation, aberrant dedifferentiation andor aberrant apoptosis of cells, comprising (i) ascertaining the level of SIP ligase expression andor SIP ligase activity in a sample of cells from a patient; and (ii) ascertaining the aggressiveness andor risk for recurrence of the disorder, at least in part, from the ascertained level of expression andor enzymatic activity, wherein an increased level in the sample, relative to a normal control sample of cells, correlates with a more aggressive form of the disorder and an increased risk of recurrence of the disorder.
60. The method of any of claims 58 or 59, wherein the CKI protein is a CIPKIP protein.
61. The method of claim 60, wherein the CIPKIP protein is p27kip1.
62. The method of any of claims 58 or 59, wherein the patient is a human.
63. The method of any of claims 58 or 59, wherein the level of CKI protein is determined by immunoassay.
64. The method of any of claims 58 or 59, wherein the level of CKI protein is determined by chromatography or electrophoresis.
65. The method of claim 58 or 59, wherein the cancer is a cancer of an epithelial tissue.
66. The method of claim 58 or 59, wherein the cancer is a cancer selected from the group consisting of carcinomas, melanomas and sarcomas.
67. The method of claim 66, wherein the cancer is a carcinoma of a tissue selected fom the group consisting of breast, ovaries, lung, intestinal, pancreas, prostate, testis, liver, skin, stomach, renal, cervical and colorectal.
68. The method of claim 58 or 59, comprising the further step of applying a treatment to the patient as determined, at least in part, on the basis of the patient’s risk of death andor recurrence of the cancer.