Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
1998-01-19
2001-03-06
McKelvey, Terry (Department: 1636)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
Reexamination Certificate
active
06197504
ABSTRACT:
Within this application, publications are referenced within parentheses. Full citations for these references may be found at the end of each series of experiments. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains.
BACKGROUND OF THE INVENTION
The
C. elegans
gene mab-21 is required for the choice of alternate cell fates by four epidermal and neuronal cells present in the lateral epidermis on each side of the male tail. Three of the four cells are products of one of nine cell sublineages leading to nine sensory rays. In mab-21 mutants, two cells, one neuronal and one glial-like, in one of the nine rays assume characteristics of the same cells found in a more anterior ray. In addition, a hypodermal cell generated by the same cell sublineage makes a different choice of fusion partner. The fourth cell affected is a hypodermal cell, which in mab-21 mutants is transformed into a neuroblast. All these cells lie together in the lateral tail epidermis, suggesting that mab-21 acts as part of a localized pattern formation mechanism. mab-21 mutant males and hermaphrodites have additional pleiotropic phenotypes affecting movement, body shape, and fecundity, suggesting that mab-21 has functions outside the tail region of males. Applicant shows that the three known alleles of mab-21 are hypomorphs of a new essential gene with an embryonic function. The mab-21 gene encodes a novel protein of 386 amino acids. In further studies of the action of mab-21 in the male tail epidermis, applicant shows by mosaic analysis that mab-21 acts cell autonomously to specify the properties of one sensory ray, but non-autonomously in the hypodermal versus neuroblast cell fate choice. Presence of cell signaling in the choice of the neuroblast fate was confirmed by cell ablation experiments. Mutations in mab-21 were shown previously to be genetic modifiers of the effects of HOM-C/Hox gene mutations on ray identity specification. The results presented here support the conclusion that mab-21 acts as part of a mechanism required for correct cell fate choice, possibly involving the function of HOM-C/Hox genes in several body regions.
During animal development, pattern formation mechanisms guide the generation of variant forms of a variety of serially repeated structures, such as segments, rhombomeres, digits, or cell sublineages. One way in which these pattern formation mechanisms exert their influence is by determining unique states of expression of HOM-C/Hox transcription factors within cells (McGinnis and Krumlauf, 1992). HOM-C/Hox transcription factors, in turn, dictate transcription of characteristic patterns of downstream target genes that determine the individual properties of separate units (Andrew and Scott, 1992; Botas, 1993).
In
C. elegans
, HOM-C/Hox genes determine variant forms of epidermal and neuronal cell sublineages that are serially repeated along the anteroposterior body axis (Sulston and Horvitz, 1977; Wang et al. 1993). As one example of this process, applicant has studied development of a set of nine bilateral pairs of peripheral sensory organs known as rays present in the posterior region of males. Each ray develops from an identical ray cell sublineage, yet each develops at a different epidermal site, can have a distinct morphology and pattern of neurotransmitter expression, and can mediate distinct behavioral responses during mating (Suiston et al., 1980; Loer and Kenyon, 1993; Liu and Sternberg, 1995). In addition, the cells of each ray appear to express a unique combination of cell recognition functions necessary for their assembly as a separate organelle. Evidence for such distinct ray morphogenetic identities came from studies of mutations in several genes required for independent development of subsets of rays (Baird et al., 1991). In males mutant for these genes, neighboring rays fuse together during development, suggesting that they have lost their distinct identities and instead express common assembly functions.
It was shown that HOM- C/Hox genes play a role in endowing separate developmental and morphological identities to the rays (Chow and Emmons, 1994). Levels of expression of these genes within the terminal cells of the ray lineages dictate distinct morphological identities that allow the rays to assemble independently. Several of the ray fusion genes applicant identified were shown to be genetic modifiers of HOM-CiHox effects on ray development, suggesting that ray fusion genes act in a common pathway with HOM-CoHox genes (Chow and Emmons, 1994). In the present paper applicant characterizes further one of these HOM-C/Hox gene modifier loci.
Mutations in the gene mab-21 resulted in the specific transformation of the identity of one of the rays into that of a more anterior ray (Baird et al., 1991). Here applicant characterizes this transformation in greater detail, and document effects of mnab-21 mutations on the cell fate choices and/or differentiation of neural and hypodermal cells. mab-21 also appears to function outside of the tail region of males, and applicant shows that his amab-21 mutations are hypomorphic alleles of a new essential gene with an embryonic function. Applicant cloned the gene and show that it encodes a novel protein of 386 amino acids. In further studies of its action in the male tail, applicant shows that mab-21 acts cell autonomously in specifying a ray identity, and thus could function together with the autonomously-acting HOM-C/Hox genes. Its action in the choice of epidermal or neuroblast cell fate by a neighboring cell was non-autonomous, however. The results are consistent with a model in which mab-21 acts as part of a localized pattern formation mechanism that dictates the fates of cells in the male tail lateral epidermis. It could as well have a wider function in specification by HOM-C/Hox genes of specialized structures or cell identities elsewhere in the body.
SUMMARY OF THE INVENTION
This invention provides an isolated polynucleotide comprising a member selected from the group consisting of: (a) a polynucleotide encoding MAB-21 protein; (b) a polynucleotide capable of hybridizing to and which is at least 60% identical to the polynucleotide of (a); and (c) a polynucleotide fragment of the polynucleotide of (a) or (b). This invention also provides a vector comprising the above-described DNA.
This invention also provides a host cell comprising the above-described vector. This invention further provides a host vector system for the production of MAB-21 protein comprising the above vector and a suitable host. In an embodiment, the suitable host is selected from a group consisting of a bacterial cell, plant and animal cell.
This invention provides a nucleic acid molecule of at least 15 nucleotides capable of specifically hybridizing with a unique sequence of a nucleic acid molecule which is complementary to the above-described nucleic acid molecule.
This invention also provides methods of detecting expression of nmab-21 or its homologs in a sample which comprises steps of: a) obtaining total mRNA from the sample; b) contacting the mRNA so obtained with a labelled nucleic acid probe molecules which are described above under hybridizing conditions; and c) determining the presence of mRNA hybridized to the molecule, and thereby detecting the expression of the mab-21 or its homologs in the sample.
This invention provides methods of detecting expression of protein products of mab-21 or mab-21 homologs in a sample which comprises steps of: a) obtaining protein extracts from the sample; b) contacting the obtained protein extract with an antibody capable of specifically recognizing the protein products of mab-21 or mab-21 homologs under conditions permitting the formation of complexes between the antibody and the protein products, measuring the amount of the formed complexes, thereby detecting the expression of the protein product of mab-21 and mab-21 homologs.
This invention also provides purified MAB-21
Cooper & Dunham LLP
McKelvey Terry
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