Chemistry: molecular biology and microbiology – Vector – per se
Reexamination Certificate
1997-12-22
2001-05-29
Kunz, Gary L. (Department: 1646)
Chemistry: molecular biology and microbiology
Vector, per se
C536S023510, C536S024100, C536S024310, C536S024330
Reexamination Certificate
active
06238916
ABSTRACT:
BACKGROUND OF THE INVENTION
The avian hypothalamus exerts a principally stimulatory influence on prolactin (PRL) secretion. Several lines of evidence support vasoactive intestinal peptide (VIP) as the most important prolactin-releasing factor (PRF) in birds. One vasoactive intestinal octacosapeptide is produced naturally in chickens and may be referred to as chicken VIP or cVIP. This particular VIP has the amino acid chain of His-Ser-Asp-Ala-Val-Phe-Thr-Asp-Asn-Tyr-Ser-Arg-Phe-Arg-Lys-Gln-Met-Ala-Val-Lys-Lys-Tyr-Leu-Asn-Ser-Val-Leu-Thr. cVIP is cross-reactive with any bird of the Avian species, including turkeys and ducks. In other words, turkeys and ducks also recognize cVIP.
VIP meets many of the qualifications of a PRF. For example, VIP stimulates PRL release from the anterior pituitary in vitro, and the response is closely correlated to the reproductive state of the animal. VIP also stimulates PRL release in vivo in median eminence-deafferentated hens (see, Opel et al.,
Proc. Soc. Exp. Biol. Med.,
187, 455 (1988)). Also, L. J. Mauro et al.,
Gen. Comp. Endoc.,
87, 481 (1992) reported the presence of high VIP concentration in median eminence, particularly the external layer. The presence of high affinity VIP receptors on the anterior pituitary cells has also been reported.
Further evidence that VIP is a PRF is provided by recent findings that lesioning of VIP cell bodies in the infundibular nuclear complex (INF) eliminates PRL increases associated with the photo-induced reproductive cycle and suppresses elevated PRL associated with incubation behavior. Finally, immunoneutralization of turkeys with endogenous VIP reduced both circulating PRL and pituitary PRL mRNA, totally blocked the PRL release effected by electrical stimulation of the medial preoptic nucleus, and blocks the hormonal and behavioral characteristics of incubating hens.
For example, the active immunization of turkey hens with VIP conjugated to an adjuvant protein was found to increase egg production. Although the bio-mechanical mechanism is not fully understood, it is believed that the antibodies so produced complex the turkey hen's naturally produced VIP. This naturally produced VIP regulates the secretion of the hormone prolactin from the pituitary gland of the turkey hen. In turn, increased prolactin secretion causes broodiness in turkey hens. Broodiness, in turn, is one of the factors that may lead to poor egg production. Accordingly, egg production is enhanced by binding the turkey hen's naturally produced VIP with the natural antibodies generated by the turkey before the turkey's VIP can act upon the turkey's pituitary gland to increase prolactin secretion.
Because of the prominent role VIP plays in the regulation of PRL secretion, it is likely that the secretion of PRL is mediated through changes in VIP secretion and/or gene expression, which may vary between the hypothalamic and hyperolactenemic birds. Therefore, a need exists to isolate, identify and regulate the structural gene encoding turkey VIP.
SUMMARY OF THE INVENTION
The present invention provides an isolated and purified DNA molecule comprising a DNA segment encoding a turkey vasoactive intestinal peptide. The present invention also provides an isolated and purified DNA molecule consisting essentially of a DNA segment encoding (a) a turkey vasoactive intestinal peptide, (b) a turkey prepro vasoactive intestinal peptide or (c) a biologically active subunit of (a) or (b). Preferably, the present invention provides a DNA segment which consists essentially of (a) DNA of SEQ ID NO:2, (b) DNA of SEQ ID NO:7, or (c) DNA of SEQ ID NO:9.
An isolated and purified DNA molecule, such as a probe or a primer, of at least seven nucleotide bases which hybridizes to these DNA molecules under the stringency conditions of Example 2, is also provided by the invention. The present invention also provides a probe or a primer comprising at least seven nucleotide bases of any of the above-disclosed single-stranded DNA sequences detectably labeled or having a binding site for a detectable label. As disclosed below, such probes or primers are useful to detect, quantify and amplify complementary DNA strands in avian tissue samples.
Thus, the present invention provides an isolated and purified DNA molecule comprising DNA encoding mature turkey vasoactive intestinal peptide (VIP) (SEQ ID NO:1), said VIP having the formula: His Ser Asp Ala Val Phe Thr Asp Asn Tyr Ser Arg Phe Arg Lys Gln Met Ala Val Lys Lys Tyr Leu Asn Ser Val Leu Thr, or a biologically-active subunit thereof. Preferably, the DNA encoding the VIP of SEQ ID NO:1 consists of the nucleotide sequence of SEQ ID NO:2: CAC TCT GAT GCT GTC TTC ACT GAC AAT TAC AGC CGC TTT CGA AAG CAA ATG GCT GTG AAG AAA TAC TTA AAC TCA GTT TTA ACT, which is also depicted, along with the amino acid sequence of VIP, in FIG.
1
.
Representative isolated and purified DNA molecules of the invention consist of: (a) the entire DNA molecule depicted in
FIG. 1
, (SEQ ID NO:7), as well as (b) a DNA molecule which encodes prepro VIP, or a biologically active subunit of mature VIP or prepro VIP. These DNA molecules are double-stranded or single-stranded, preferably, they are cDNA. For example, the native DNA molecule encoding turkey prepro VIP of SEQ ID NO:8 is depicted under amino acids residues 1-165, and is designated SEQ ID NO:7.
Also encompassed by the invention is an isolated and purified DNA molecule encoding mature turkey VIP which hybridizes to a DNA sequence complimentary to DNA of SEQ ID NO:5 under the high-stringency hybridization conditions disclosed herein below, or under conditions of greater stringency.
As used herein, the terms “isolated and purified” refer to in vitro isolation of a DNA molecule or peptide from its natural cellular environment, and from association with other coding regions of the avian genome, so that it can be sequenced, replicated and/or expressed.
Preferably, the isolated and purified DNA sequences of the invention comprise a single coding region, and are no more than about 500-900 base pairs in length. Thus, the present DNA molecules are those “consisting essentially of” or those consisting of DNA segments encoding VIP, pre-pro VIP or a biologically active subunit of either. Unexpectedly, certain DNA molecules of the invention were found to include a DNA segment encoding a peptide known as peptide histidine isoleucine (PHI), which is also within the scope of the term “turkey vasoactive intestinal peptide.”
The term “biologically active” refers to a polypeptide which has at least about 50% of the in vivo biological activity of turkey VIP of SEQ ID NO:1, as determined by the assays disclosed in commonly-assigned U.S. Pat. No. 5,557,033 filed Sep. 22, 1992.
The present invention also provides an expression vector, preferably a linear vector, comprising an isolated DNA molecule encoding (a) turkey vasoactive intestinal peptide (VIP), said VIP having the formula of SEQ ID NO:1; (b) prepro VIP of SEQ ID NO:8; or (c) a biologically active subunit peptide of (a) or (b). Preferably, the vector comprises a single coding region, and comprises a second DNA sequence operably linked to the coding sequence, and capable of directing expression of the VIP polypeptide of (a)-(c), such as a promoter region operably linked to the 5′ end of the coding DNA sequence. Such expression vectors can be constructed and employed to transform host cells, i.e., procaryotic cells, in order to produce preselected VIP peptides. Although the present vectors contain only one VIP coding region, they also can contain a DNA sequence which is a selectable marker gene or reporter gene, as described below. The expression vectors can also be constructed and employed to transform eukaryotic cells. The present invention also provides a transformed eukaryotic host cell, which host cell contains an exogenous avian vasoactive intestinal peptide gene comprising: a native vasoactive intestinal peptide gene; and a DNA molecule encoding an exogenous avian vasoactive intestinal peptide wherein the DNA molecule expresses the ex
Gucker Stephen
Kunz Gary L.
Regents of the University of Minnesota
Schwegman Lundberg Woessner & Kluth P.A.
LandOfFree
DNA encoding turkey hypothalamic vasoactive intestinal peptide does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with DNA encoding turkey hypothalamic vasoactive intestinal peptide, we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and DNA encoding turkey hypothalamic vasoactive intestinal peptide will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2533353