US20130089867A1

US20130089867A1 - Isoforms of the human sst5 receptor originated by alternative splicing and oligonucleotide pairs to detect them by pcr

Info

Publication number: US20130089867A1
Application number: US13/713,058
Authority: US
Inventors: Mario Durán Prado; Antonio Jesús Martínez Fuentes; Rafael Vazquez Martínez; Socorro García Navarro; María del Mar Malagón Poyato; Justo Pastor Castaño Fuentes; Francisco Gracia-Navarro
Original assignee: Universidad de Cordoba
Current assignee: Universidad de Cordoba
Priority date: 2007-10-17
Filing date: 2012-12-13
Publication date: 2013-04-11
Also published as: DK2216340T3; EP2216340A1; ES2401233T3; US8354273B2; US20140248628A1; WO2009050309A1; US20120003252A1; EP2216340A4; JP2011500047A; EP2216340B1; PL2216340T3; PT2216340E

Abstract

The present invention is referred to two human nucleic acids that comprise sequences encoding two new isoforms of the human somatostatin receptor type 5 originated by alternative splicing, named sst5B and sst5C, with possible involvement in tumor processes. In addition, the invention is referred to oligonucleotide pairs allowing their differential detection in several tissues using the PCR technique.

Description

This application is a divisional application of U.S. patent application Ser. No. 12/738,131, filed May 31, 2011, now allowed, based upon the United States national stage filing under 35 U.S.C. §371 of International (PCT) Application Number PCT/ES07/00627 filed Oct. 27 2007, and designating the US.

INVENTION The present invention is referred to two new isoforms of the human somatostatin receptor 5, as well as to their detection in biological samples.

BACKGROUND OF THE INVENTION

The hypothalamic neuropeptide somatostatin (SRIF) acts on several organs and tissues widespread distributed in the organism, exerting mainly an inhibitory effect on hormone secretion, as well as on other biological processes (Moller et al., 2003).
These inhibitory, but sometimes stimulatory effects (Castaño et al., 1996) are exerted through a family of seven transmembrane domains receptors (7TMDs) coupled two G proteins (GPCRs), called somatostatin receptors or ssts. The ssts share a common structure consisting in an extracellular amino terminal domain, connected to seven hydrophobic domains inserted into the membrane, which are connected by eight hydrophilic segments ending in an intracellular carboxyl terminal domain, this latter important in the modulation of second messengers pathways.
To date, in mammals, there are five different sst subtypes, from sst1 to sst5, and additionally, in rat and mouse, two isoforms of the subtype 2 (sst2A and sst2B) produced by alternative splicing of the precursor mRNA which encode two proteins differing at their intracellular carboxy terminal region and that possess a different ability to regulate second messengers pathways. However, in fish, there have been described other isoforms of each sst subtype, but due to duplication events instead of alternative splicing.
GPCRs, and among them the ssts, are involved in many cellular processes of high clinical relevance, mediated by signal transduction pathways depending on G proteins coupling. More specifically, one of these sst subtypes, the human sst5 (WO 0177172, WO 0155319, WO 0136446, EP 1369698, WO 03104816) has been linked, in mammals, with many pathologies as hematological diseases, cardiovascular diseases, alterations of the central and peripheral nervous systems, cancer, inflammatory diseases, hepatic diseases, gastrointestinal and urinary diseases, etc. (WO03104816).
The human somatostatin receptor type 5, sst5, is deposited in public databases with accession numbers G139756975, G121954086, G113937340. These sequences contain the cDNA corresponding to the coding sequence as well as the complete genetic structure of the receptor, including the promoter sequence, introns and the 5′ and 3′ untranslated sequences. To date, there has not been described an alternative splicing of the mRNA that originates an alternative isoform different to the deposited in the databases and widely described in bibliography.
Recently, it has been cloned the sequence corresponding to the mRNA containing the CDS of the porcine sst5, as well as the 5′ and 3′ non coding regions (Duran et al., 2005; publication in preparation). During the cloning by RACE PCR, two partial and latter complete variants of the mRNA were obtained which, by alternative splicing, encode two new isoforms of the receptor, similar to that reported for the murine sst2, but in this instance, encoding receptors of six and three transmembrane domains, named porcine isoforms sst5B and sst5C (p-sst5B and psst5C) respectively.
There are data of truncated GPCRs originated by alternative splicing of the mRNA, that encode proteins with less than seven transmembrane domains, as described previously for the GHRH receptor (Rekasi et al., 2000), GnRHR (Pawson et al., 2005), prostaglandin receptor (Ishii et al., 2001), etc., being some of them functional and having possible relevance in tumor processes.
After the results obtained for the porcine sst5, it began the cloning by RACE PCR of the putative human homologues of the sst5B and sst5C isoforms, to further evaluate their presence and importance in endocrine tumors, using the PCR technique.

BIBLIOGRAPHY CITED IN THE TEXT

- 1. Moller L N, Stidsen C E, Hartmann B, Holst J J. 2003. Somatostatin receptors. Biochemica et Biophysica Acta, 1616: 1-84.
- 2. Castaño J P, Torronteras R, Ramirez J L, Gribouval A, Sánchez-Hormigo A, Ruiz-Navarro A, Gracia-Navarro F. 1996. Somatostatin increases growth hormone (GH) secretion in a subpopulation of porcine somatotropes: evidence for functional and morphological heterogeneity among porcine GH-producing cells. Endocrinology, 137:129-136.
- 3. Rekasi Z, Czompoly T, Schally A V, Halmos G. 2000. Isolation and sequencing of cDNAs for splice variants of growth hormone-releasing hormone receptors from human cancers. PNAS, 97: 10561-10566.
- 4. Pawson A J, Maudsley S, Morgan K, Davidson L, Naor Z, Millar R. 2005. Inhibition of human type I gonadotropin-releasing hormone receptor (GnRHR-I) function by expression of a human type II GnRHR gene fragment. Endocrinology. 146(6):2639-2649.
- 5. Ishii Y, Sakamoto K. 2001. Suppression of protein kinase C signaling by the novel isoform for bovine PGF2a Receptor 1. Biochemical and Biophysical Research Communications, 285: 1-8.
- 6. Landa R L, Harbeck M, Kaihara K, Chepurny O, Kitiphongspattana K, Graf O, Nikolaev V O, Lohse M J, Holz G G, Roe M W. 2005. Interplay of Ca²⁺ and cAMP signaling in the insulin secreting MING β-cell line. Journal of Biological Chemistry, 2; 280(35):31294-31302.
- 7. Vilardaga J P, Bunemann M, Krasel C, Castro M & Lohse M J. 2003. Measurement of the millisecond activation switch of G protein-coupled receptors in living cells. Nat Biotechnol 21 807-812.

DESCRIPTION OF THE INVENTION

For the correct interpretation of the present text the following concepts are detailed:
A “somatostatin receptor” is a transmembrane protein coupled to a G protein, belonging to the family of seven transmembrane domains, which is activated by the hypothalamic peptide somatostatin.
“RACE PCR” is referred to Random Amplification of cDNA Ends. It is a PCR (Polymerase Chain Reaction) based technique that allow the introduction of known oligonucleotide sequences into unknown cDNA sequences, that are used as target to amplify by PCR the cDNA sequence comprised between the mentioned oligonucleotides and the region of interest.
“Pituitary Cushing” is referred to the “cushing” syndrome or hypercortisolism. It is a disease caused by an increase of cortisol synthesis or by the excessive use of this or other steroid hormones. A pituitary “cushing” is when the disease is due to an increase of the production of the pituitary adrenocorticotroph hormone.
The present invention comprises the determination of the DNA sequence encoding two new isoforms of the somatostatin receptor type 5 (sst5), named sst5B and sst5C, of five and four transmembrane domains respectively, produced by alternative splicing of the mRNA contained into the genomic sequence deposited in the database with accession number GI13937340 (FIG. 1).
With the procedure described in the way to carry out the invention, it is possible to obtain recombinant DNA molecules encoding polypeptides that show, at least in part of the sequence, structural motifs of somatostatin receptors.
The invention is also referred to polynucleotide DNA sequences that hybridize, under restrictive conditions, with those of the new isoforms, which implies a homology level of at least 60% between their nucleotide sequences, preferably a homology of 75% and more preferably a homology of 90%, or sequences derived from them by variation of the genetic code or by mutagenesis.
The procedure used in the invention allows the obtaining of recombinant functional polypeptides for their further study. The recombinant DNA molecules as the described in SEQ ID 1, SEQ ID 3, SEQ ID 5 and SEQ ID 7 or derived from those are inserted into expression vectors. Both polypeptides expressed into host cells offer a new screening system to test new drugs and ligands able to bind selectively in vivo and in vitro to the sst5B and sst5C isoforms, as well as systems to study the modulation of second messenger pathways by each isoform in response to drugs.
The invention object of this application is referred to a purified human nucleic acid that encodes an isoform of the human somatostatin receptor type 5 (sst5) chosen between: sst5B (SEQ ID 5), sst5C (SEQ ID 7), their complementary sequence, a sequence with a 90% homology, and fragments of them.
The invention is also referred to a purified human nucleic acid characterized because it comprises a partial coding sequence contained in SEQ ID 1 and SEQ ID 3.
In a specific consecution, the invention is referred to a human nucleic acid characterized because it contains the 3′ RACE fragment corresponding to the sst5B which sequence is SEQ ID 1, or its partial fragments. In another particular consecution, the invention is referred to a human nucleic acid characterized because it contains the 3′ RACE fragment corresponding to sst5C which sequence is SEQ ID 3, or partial fragments derived from it.
In a preferable consecution, the invention is referred to a purified polypeptide characterized because its amino acid sequence is defined in SEQ ID 2, SEQ ID 4, SEQ ID 6 and SEQ ID 8, and is encoded by one of the oligonucleotides described before in the text.
In second thoughts, the invention is referred to an expression vector characterized because it comprises a nucleotide sequence described before, transcriptionally coupled to an exogenous promoter. In a specific consecution, the mentioned expression vector is characterized because its nucleotide sequence encodes a polypeptide like the defined previously in the text.
In a specific consecution, the methods described previously are characterized because are performed in vitro. In a preferable consecution, the searching is performed in whole cells. In a preferable consecution, the mentioned method is characterized because the polypeptides detailed in SEQ ID 2, SEQ ID 4, SEQ ID 6 and SEQ ID 8, come from an expression vector defined previously in the text. In a more preferable consecution, the mentioned polypeptide corresponds to one of the encoded by SEQ ID 1, SEQ ID 3, SEQ ID 5, SEQ ID 7, or their fragments.
On the other hand, the invention is also referred to new oligonucleotide pairs detailed in the sequences SEQ ID 9, SEQ ID 10, SEQ ID 11, SEQ ID 12, SEQ ID 13, and SEQ ID 14 or sequences homologues in at least 90%, that allow the amplification by PCR of the isoforms A, B and C of the human sst5. In a specific consecution, the invention is referred to the use of these oligonucleotides to amplify selectively the isoforms sst5A, sst5B and sst5C, with any PCR variant. In a preferable consecution, the invention is also referred to the use of the oligonucleotides to study the quantitative tissue distribution of sst5A, sst5B and sst5C in normal and tumor tissues.
In a specific consecution, the invention is referred to a cDNA characterized because it hybridizes with the total or partial sequences contained in SEQ ID 1, SEQ ID 3, SEQ ID 5 or SEQ ID 7.
On the other hand, the invention is also referred to a cDNA contained in SEQ ID 1, SEQ ID 3, SEQ ID 5, SEQ ID or sequences with at least a 90% homology, characterized because it is able to silence independently or together, the genetic expression of the sst5B and sst5C isoforms.
A specific consecution of the present invention is referred to the use of sequences contained in SEQ ID 1, SEQ ID 3, SEQ ID 5 or SEQ ID 7 to generate selective antibodies that distinguish between the sst5B and sst5C isoforms.
The present invention allows the developement of new drugs able to bind selectively to the new sst5 isoforms, sst5B and sst5C, acting as agonists, antagonists or inverse agonists, using second messenger measurement techniques as the microfluorimetric measurement of intracellular calcium (Landa et al., 2005).
More specifically, the insertion of the recombinant DNA, contained in SEQ ID 1, SEQ ID 3, SEQ ID 5 and SEQ ID 7 or derived from them, in eukaryotic expression vectors pCDNA3 like (Invitrogen) allows the transfection of those constructs in tumor cell lines as CHO-K1 and HEK-293T, widely used in the study of other somatostatin receptor subtypes. The process, which methodology is detailed in Landa et al., (Landa et al., 2005) is outlined as follows:

- (1) Culture of the cell line of interest onto sterile glass coverslips.
- (2) Transfection of the cell line with the appropriate recombinant plasmid.
- (3) Incubation of the transfected cells for 30 min at 37° C. with 2.5 μM Fura-2 AM (Molecular Probes, Eugene) in phenol free DMEM supplemented with 20 mM NaHCO₃at pH 7.4.
- (4) Assembly of the coverslip into a chamber coupled to the stage of an inverted microscope Nikon Eclipse TE 2000 E, coupled to a Hamamatsu CCD digital camera (Hamamatsu Photonics, Hamamatsu), both under the control of MetaFluor software (Molecular Devices).
- (5) Analysis of the transfected cells with a 40× oil immersion objective, with a dual and alternating sample excitation at 340 and 380 nm and measurement at 505 nm at 5 sec intervals.
- (6) Changes in the intracellular Ca²⁺ concentration before and after the drug administration are analyzed with the MetaFluor software as ratio of the intensity obtained from the images at both excitation wavelengths, 340 and 380 nm.

The present invention makes posible the developement of new drugs that modify the basal state of the new somatostatin 5 isoforms, sst5B and sst5C. Therefore, the present invention will allow using FRET (Fluorescence Resonance Energy Transfer) technologies to measure the physical interaction of the sst5B and sst5C isoforms with themselves and with other proteins belonging to the GPCR family. With this technique it is possible to study, in a rapid and accurate way, changes in the basal state of the receptor, whether they are due to aggregation or dissociation of ternary protein complexes, in response to a drug. More specifically, the insertion of the recombinant DNA molecules contained in SEQ ID 1, SEQ ID 3, SEQ ID 5 and SEQ ID 7, or derived from them, in eukaryotic expression vectors variants E-GFPN1 like (Clontech), as E-CFPN1 and E-YFPN1 will allow the cotransfection of these recombinant constructs in tumor cell lines like CHO-K1 or HEK-293T. The process, which methodology is detailed in Vilardaga et al., (Vilardaga et al., 2003) is outlined as follows:

- (1) Culture of the cell line of interest onto sterile glass coverslips.
- (2) Cotransfection of the cell line with the plasmid pair of interest.
- (3) Assembly of the coverslip into a chamber coupled to the stage of an inverted microscope, as the Nikon Eclipse TE 2000 E, coupled to a Hamamatsu CCD digital camera (Hamamatsu Photonics, Hamamatsu), both under the control of MetaFluor software (Molecular Devices).
- (4) Analysis of the transfected cells with a 40× oil immersion objective, with a dual and alternating sample excitation at 440 and 495 nm and measurement of the emission signal at 510 and 540 nm respectively, at 5 sec intervals.
- (5) Changes in the intensity of both fluorescent proteins, E-CFP and E-YFP, before and after the drug administration are analyzed with the MetaFluor software as ratio of the intensity obtained from the images at both emission wavelengths, 510 and 540 nm.

DESPCRIPTION OF THE FIGURES

FIG. 1: Schematic representation of the partial sequences corresponding to the sst5B and sst5C isoforms using the 3′ RACE PCR technique. They are represented the steps described in the text and shown the oligonucleotides used in each instance, with the nomenclature indicated in Table 1.

FIG. 2: Schematic representation of the amplification of sst5B and sst5C coding sequences using PCR and triple ligation. They are represented the steps described in the text and shown the oligonucleotides used in each instance, with the nomenclature indicated in Table 1.

WAY TO CARRY OUT THE INVENTION

Hereafter it is described an example for a better understanding of the invention.

EXAMPLE

To clone the partial sequences of the sst5B and sst5C isoforms, the subsequent steps described in FIG. 1 were followed:

- (1) Isolation of total RNA from several tissues, followed by its retrotranscription.
- (2) Amplification of the 3′ region of the sst5B and sst5C isoforms using RACE PCR; and
- (3) reamplification using nested oligonucleotides.
- (4) Cloning of the PCR products of step (3) and sequencing to determine their correct sequence.
- (5) Verification of the transcription origin of the sst5B and sst5C isoforms by PCR using as template the cDNA fragments obtained in (1).
- (6) Reamplification of (5) to check the specificity of the PCR bands obtained in that step.

The amplification method described was carried out following the indications of the “GeneRacer® kit”, from Invitrogen® life technologies, with the exception of steps (5) and (6).
To clone the coding sequences and for the functional expression of the isoforms sst5B and sst5C, it was used the strategy outlined in FIG. 2:

- (1) The exon 1 (E1) as well as the exon 2 (E2) corresponding to each isoform were amplified from human genomic DNA.
- (2) Enzymatic digestion of the PCR fragments, and (3) Ligation of E1 and E2 between them and into an expression vector (not shown in the scheme).

Also, they were designed oligonucleotide pairs able to discriminate among the isoforms sst5A, sst5B and sst5C (SEQ ID 9 and SEQ ID 14) and that can be used with quantitative aims, amplifying selectively each isoform with the PCR conditions detailed subsequently in the text.
Nucleic Acids Isolation.
RNA isolation. It was performed using the Trizol reagent Invitrogen®, according the suppliers recommendations. Tissues corresponding to two pituitary adenomas diagnosed as non functioning and “cushing” were used as starting material for total RNA extraction. The resulting RNA was resuspended in 12 μl DEPC treated H₂O and 1 μl was used for spectrophotometric quantification. For the retrotranscription, 2 μg of ARN from each sample were used in a 20 μl final volume. In addition, RNA from HeLa cells was used, in this instance supplied with the “GeneRacer kit”, using the same amount of RNA for the retrotranscription reaction (FIG. 1.1.). The retrotranscription reactions were carried out following the recommendations of the “GeneRacer® kit” from Invitrogen®. For diagnostic purposes, total RNA was extracted from 15-100 mg tissue of a heterogeneous tumor pituitary panel. The resulting RNA was also resuspended in a 12 μl DEPC treated H₂O final volume, of which one was used for spectrophotometric quantification. Between 2 and 5 μg of RNA were used for the retrotranscription reaction in a final volume of 20 μl, this time using the “Powerscript” (BD Biosciences) retrotranscriptase and following the manufacturer's protocol.
Genomic DNA isolation. DNA was isolated from 10⁷human lymphocytes as starting material, using the Trizol reagent. The genomic DNA obtained was quantified spectrophotometrically.
PCR Amplification and Obtaining of Partial Sequences of the sst5B and sst5C Isoforms.
As indicated previously, the amplification was carried out using the “GeneRacer® kit” from Invitrogen® combined with oligonucleotides specifically designed, specified in Table 1.

TABLE 1

Oligonucleotides used for the selective
amplification of h-sst5B and h-sst5C partial sequences.

	Sequence 5′•3′, position in
Name	reference sequence and amino acids		Reference
(SEQ ID)	sequence	Way	sequence

Hum_sst5_ATG	1-ATGGAGCCCCTGTTCCCAGCCT-22	Sense	GI39756975
(15)	1-MEPLFPA-7

Ra_hum_sst5_3′	490-TGGGTCCTGTCTCTGTGCATGTC-512	Sense	GI39756975
(16)	164-WVLSLCM-170

Ra_hum_sst5_3′	523-CTGGTGTTCGCGGACGTGCAG-543	Sense	GI39756975
N (17)	175-LVFADVQ-181

sst5B-C_E1_U_	1-TCAAGCTTCGATGGAGCCCCTGTTCCCAGC-20	Sense	GI39756975
HindIII (18)	1-MEPLFP-6

sst5B-E1_L	599-CGGCGCGAAGAAGCCCAGCAC-619	Antisense	GI39756975
blunt (19)	207-VLGFFAP-213

sst5B-E2-U	2275-CTGCTGAGAGGCAGCGGCC-2293	Sense	GI13937340
blunt(20)	LLRGSG

sst5B-E2-	2437-	Antisense	GI13937340
L_BamHI (21)	TTAGGATCCTCAGAGCAAGGCCAAGTTGCC-2457
	GNLALL

sst5C-E1_L	675-GTTGCAGGTACCGCCCTCCTG-695	Antisense	GI39756975
blunt (22)	181-QEGGTCN-187

sst5C-E2_U	2548-CGTCTGCCCAGAGCAGGACCTC-2569	Sense	GI13937340
blunt (23)	RLPRAGP

sst5C-	2617-	Sense	GI13937340
E2_L_BamHI	ACTGGATCCTCAGCCTGGGCCTTTCTCCTG-2637
(24)	QEKGPG

*The bases in italics represent cutting sites for restriction enzymes.

After the retrotranscription reaction, 100 ng of cDNA were used for each PCR, using the oligonucleotides Ra_hum_sst5 _—3′ (SEQ ID 16) and GeneRacer 3′ (FIG. 1.3.), with a program consisting in an initial denaturation of 2 minutes at 94° C., followed by five repeats of a 30 seconds denaturation at 94° C., 1 minute 30 seconds of annealing and extension at 72° C. and another 35 cycles similar to the previous, but employing a less stringent annealing temperature of 66° C. The amplification program continued with a final extension of minutes at 72° C. to finish the incomplete PCR fragments.
One μl of each PCR product was reamplified with the nested oligonucleotides Ra_hum_sst5 _—3′N (SEQ ID 17) and GeneRacer 3′N (FIG. 1.4.) with a program consisting in an initial denaturation of 2 minutes at 94° C., followed by thirty two cycles of a 30 seconds denaturation at 94° C., 30 seconds of annealing at 66° C. and 40 seconds of extension at 72° C. The amplification program continued with a final extension of 7 minutes at 72° C. to end the incomplete PCR fragments.
Both amplifications were performed with the Certamp (BioTools, Spain) polimerase mixture supplied with a specific buffer for complex amplifications. The different PCR reactions were carried out with all the cDNA in parallel. PCR products were visualized in a 1% agarose gels and the bands of interest were purified with the QuiaQuick Mini Elute kit (Quiagen). The purified blunted ends PCR products were cloned into the EcoRV site of the pBluescript KSII+ plasmid and then sequenced, resulting in the sequence of 609 base pairs (SEQ ID 1) obtained from cDNA coming from HeLa RNA and another sequence of 257 base pairs (SEQ ID 3), obtained from the cDNA coming from the pituitary “cushing”. Both cDNA obtained from HeLa and the pituitary “cushing” were further amplified with the oligonucleotides Hum_sst5_ATG (SEQ ID 15) and GeneRacer 3′ (FIG. 1.5.), and the products obtained were reamplified with the same oligonucleotide Hum_sst5_ATG and the nested oligonucleotide GeneRacer 3′N (FIG. 1.6.). Both PCR reactions were carried out with the same program consisting in an initial denaturation of minutes at 94° C., followed by forty cycles of a 30 seconds denaturation at 94° C., 30 seconds of annealing at 62° C. and 1 minute and 30 seconds of extension at 72° C. The amplification program continued with a final extension of 7 minutes at 72° C. to end the incomplete PCR fragments. The visualization in a 1% agarose gel allowed checking the presence of a 1099 base pairs band obtained from HeLa cDNA and another 747 base pairs band obtained from the pituitary “cushing” cDNA. These results showed that similarly to the porcine truncated isoforms, both truncated isoforms sst5B and sst5C share the same putative translational start with the long isoform, sst5A (accession number GI39756975).
Obtaining of the Coding Sequence Corresponding to the sst5B and sst5C Isoforms.
For the cloning and functional expression of the human sst5B and sst5C isoforms, it was used a strategy based in the independent amplification of the two exons that constitute each isoform (FIG. 2) and a further ligation of both fragments into a eukaryotic expression vector. More in detail, using genomic DNA as template, the E1 of each isoform was amplified with a common sense oligonucleotide for sst5B and sst5C, sst5B-C_E1_U_HindIII (SEQ ID 18) that incorporates a restriction sequence for the HindIII enzyme, and a specific antisense oligonucleotide for each isoform, sst5B-E1_L_blunt (SEQ ID 19) and sst5C-E1_L_blunt (SEQ ID 22) for sst5B and sst5C, respectively (FIGS. 2.1. and 3.2.). The E2 were similarly amplified, with the sense and antisense specific oligonucleotide pair sst5B-E2-U blunt (SEQ ID 20)/sst5B-E2-L_BamHI (SEQ ID 21) for the sst5B isoform, and sst5C-E2_U_blunt (SEQ ID 23)/sst5C-E2_L_BamHI (SEQ ID 24) for the sst5C isoform. The four PCR reactions were performed in parallel with a program consisting in an initial denaturation of 2 minutes at 94° C., followed by thirty four cycles of a 30 seconds denaturation at 94° C., seconds of annealing at 62° C. and 40 seconds of extension at 72° C. In all instances it was used a high fidelity polymerase like Pfu Ultra (Stratagene), supplementing the reaction with 1M betaine (Sigma). With these reactions, it was able to introduce a HindIII cutting site in the 5′ of each El, a blunt end in the 3′ of the E1, a blunt end in the 5′ of the E2 and a BamHI cutting site in the 3′ of each E2. It was previously checked that none of these enzyme cut into the target sequences. The PCR fragments obtained were purified with the QuiaQuick Mini Elute kit (Quiagen) and after enzymatic digestion with HindIII and BamHI, were triple ligated into the eukaryotic expression vector pCDNA3+ (Invitrogen) previously linearized with the same restriction enzymes. Both constructs, sst5B-pCDNA3+ and sst5C-pCNA3+, were sequenced at least twice, to check the integrity of the sequences and to compare them with the genomic sequence GI13937340, that contains both isoforms, using the program BLAST 2 SEQUENCES http://www.ncbi.nlm.nih.gov/blast/b12seq/wblast2.cgi.
Selective Amplification with Quantitative Aims of Partial Sequences Corresponding to the sst5A, sst5B and sst5C Isoforms.
Oligonucleotide pairs with a diagnostic aim were developed allowing the selective discrimination by PCR of each of the human sst5 isoforms, sst5A (GI39756975), sst5B and sst5C. The oligonucleotide pair Hum_sst5A_cuant_U/Hum_sst5A_cuant_L, SEQ ID 9 and SEQ ID 10, respectively, amplifies a PCR product of 154 base pairs, using an annealing temperature of 68° C. The oligonucleotide pair Hum_sst5B_cuant_U/Hum_sst5B_cuant_L, SEQ ID 11 and SEQ ID 12, respectively, at an annealing temperature of 68° C., amplifies a PCR product of 142 base pairs contained into the sequence corresponding to the sst5B (SEQ ID 5) and does not amplify the isoforms sst5C or sst5A (GI39756975) while it amplifies a 1643 PCR fragment contained into the human genomic sequence GI13937340 which includes the intron located between the exons E1 and E2 of the sst5B isoform. The oligonucleotide pair Hum_sst5C_cuant_U/Hum_sst5C_cuant_L, SEQ ID 13 and SEQ ID 14, respectively, at an annealing temperature of 68° C., amplifies a PCR fragment of 137 base pairs contained into the sequence corresponding to the sst5C (SEQ ID 6), and also amplifies a 488 base pair fragment contained into the sequence corresponding to the sst5B, and additionally amplifies a 1989 base pairs fragment contained into the human genomic sequence GI13937340 which includes the intron located between the exons E1 and E2 of the sst5C isoform. The three PCR reactions were carried out in parallel using a PCR program consisting in an initial denaturation of 2 minutes at 94° C., followed by thirty seven cycles of a 10 seconds denaturation at 94° C., 10 seconds of annealing at 68° C. and 10 seconds of extension at 72° C. With these PCR settings, each oligonucleotide pair only amplifies selectively a specific isoform, avoiding the additional PCR fragments mentioned above. In all instances, the PCR reactions were supplemented with 1M betaine (Sigma). This methodology allowed screening the presence of the sst5B and sst5C isoforms in several pituitary tumors of different etiology.

Claims

1. Purified somatostatin receptor type 5 oligonucleotides, fragments, homologs, or fully complementary nucleic acids thereof, wherein

a) said purified oligonucleotide, fragment, homolog or fully complementary nucleic acid comprises a sequence having at least 90% identity with SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14, and

b) said purified oligonucleotide, fragment, homolog or fully complementary nucleic acid is capable as acting as a primer for the PCR amplification of a human somatostatin receptor type 5 nucleic acid.

2. The purified oligonucleotides, fragments, homologs, or fully complementary nucleic acids according to claim 1, wherein said human somatostatin receptor type 5 nucleic acid comprises a sequence having at least 90% identity with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.

3. The purified oligonucleotides, fragments, homologs, or fully complementary nucleic acids according to claim 2, wherein said human somatostatin receptor type 5 nucleic acid is SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.

4. The purified oligonucleotides, fragments, homologs, or fully complementary nucleic acids according to claim 3, wherein said purified oligonucleotide, fragment, homolog or complementary nucleic acid is SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14.

5. A method of selectively amplifying a human somatostatin receptor type 5 nucleic acid, wherein purified oligonucleotides, fragments, homologs, or fully complementary nucleic acids thereof comprising a sequence having at least 90% identity with SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14, are used for the PCR amplification of a human somatostatin receptor type 5 nucleic acid.

6. A method according to claim 5, wherein said amplified human somatostatin receptor type 5 nucleic acid comprises a sequence having at least 90% identity with SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.

7. A method according to claim 6, wherein said amplified human somatostatin receptor type 5 nucleic acid is SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.

8. A method according to claim 5, wherein said purified oligonucleotides, fragments, homologs, or fully complementary nucleic acids thereof are selected from the group consisting of SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14, and wherein said amplified human somatostatin receptor type 5 nucleic acid is SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.

9. A method of determining the tissue distribution of human somatostatin receptor type 5 nucleic acids utilizing purified oligonucleotides comprising a sequence having at least 90% identity with SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13 or SEQ ID NO:14, or purified fragments, homologs, or fully complementary nucleic acids thereof.

10. A method of gene silencing the expression of either or both sst5B or sst5C somatostatin receptor type 5 genes, said method comprising administering a somatostatin receptor type 5 nucleic acid, fragment, homolog or fully complementary nucleic acid comprising a sequence having at least 90% homology to SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5 or SEQ ID NO:7.

11. The use of a somatostatin receptor type 5 polypeptide, fragment or homolog thereof for the production of antibodies, wherein

a) said somatostatin receptor type 5 polypeptide, fragment or homolog shares at least 90% identity with SEQ ID NO:2, SEQ ID NO:4, SEQ ID NO:6 or SEQ ID NO:8, and wherein

b) said antibodies discriminate sst5B and sst5C polypeptide isoforms.