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PubMed2000

Newcastle disease virus phosphoprotein gene analysis and transcriptional editing in avian cells.

Nucleotide sequence was determined for the phosphoprotein (P) gene from 23 Newcastle disease virus (NDV) isolates representing all defined pathotypes with different chronological and geographic origins. Sequence variation, with synonymous substitutions dominating, occurred throughout the P gene. An exception was a conserved central region containing the transcriptional editing site. Four G nucleotide additions were detected in NDV P gene mRNA potentially creating alternative open reading frames. However, only one in-frame stop codon exists with a single G addition among all isolates that would allow for a potential V protein. A second potential stop codon does not exist in the P gene consensus sequence among all isolates with more than one G nucleotide addition at the editing site. This precludes a possible W protein in these isolates. A second potential alternative in-frame start site exists among all isolates that could encode a predicted X protein for NDV. Comparison of the P gene editing sites among the Paramyxovirinae and predicted P gene usage demonstrates that NDV more closely resembles the respiroviruses and morbilliviruses. Phylogenetic analysis of P gene sequences among NDV isolates demonstrates there are two clades of these viruses. One group includes viruses isolated in the US prior to 1970, while a second cluster includes virulent viruses circulating worldwide.

Amino Acid Sequence↗
PubMed2001

Transcriptional complexity of the Anopheles stephensi nitric oxide synthase gene.

Anopheles stephensi nitric oxide synthase (AsNOS) is a single copy gene that shares significant structural homology with the three human NOS genes and is inducibly expressed in Plasmodium-infected mosquitoes. Exon-specific Northern analyses and exon-spanning polymerase chain reaction amplification were used to further characterize transcription from this gene. A total of 18-22 AsNOS transcripts, ranging in size from 1.0 to 7.5 kb, were detected in replicated Northern blots from three separate cohorts of mosquitoes. Three transcripts (1604, 2330, and 2585 bp) were significantly induced in Plasmodium-infected mosquitoes (p<0.05), while others showed varying patterns of induction or downregulation. Five splice variants contained deletions of 1-7 exons. All but one deletion pattern was predicted to introduce in-frame stop codons or alter the translational reading frame. A novel insertion derived from intron sequence was predicted to introduce in-frame stop codons following exon 11. Two truncated novel exon 1 variants were identified that are homologous to a previously published 5' sequence for this exon. The large number of AsNOS transcripts and diversity in AsNOS splicing and exon 1 sequences indicate that transcriptional complexity is a hallmark of both invertebrate and vertebrate NOS genes.

Alternative Splicing↗
PubMed1987

Specific occurrence of selenium in enzymes and amino acid tRNAs.

In contrast to the widespread ability of bacteria, plants, and animals to incorporate selenium nonspecifically into proteins in the form of selenomethionine residues, the selenoamino acid selenocysteine occurs as a highly specific component of a few selenium-dependent enzymes. Selenocysteine has been identified in glycine reductase, formate dehydrogenase, and hydrogenase of bacterial origin and glutathione peroxidase from mammalian and avian sources. In these enzymes there is evidence that the selenol group, which is largely ionized at physiological pH, functions as a redox center. It now seems clear, from studies with both prokaryotes and eukaryotes, that the UGA opal stop codon is used to specify the cotranslational insertion of selenocysteine into proteins. The factors that allow this unusual use of the stop codon are, however, unknown. The occurrence of selenium as a normal constituent of several bacterial tRNA species has been established. The presence of a selenonucleoside, 5-methylaminomethyl-2-selenouridine, in the first or wobble position of the anticodons of certain glutamate and lysine iso-acceptor species influences codon-anticodon interaction and thus may serve to regulate translational processes. The biosynthesis of the selenonucleoside appears to involve the ATP-dependent activation of the sulfur in a preformed 5-methylaminomethyl-2-thiouridine residue in tRNA and replacement of the sulfur with selenium.

Animals↗
PubMed2005

Hepatitis C virus F protein sequence reveals a lack of functional constraints and a variable pattern of amino acid substitution.

Hepatitis C virus (HCV) is an important human pathogen that affects 170 million people worldwide. The HCV genome is an RNA molecule that is approximately 9.6 kb in length and encodes a polyprotein that is cleaved proteolytically to generate at least 10 mature viral proteins. Recently, a new HCV protein named F has been described, which is synthesized as a result of a ribosomal frameshift. Little is known about the biological properties of this protein, but the possibility that the F protein may participate in HCV morphology or replication has been raised. In this work, the presence of functional constraints in the F protein was investigated. It was found that the rate of amino acid substitutions along the F protein was significantly higher than the rate of synonymous substitutions, and comparisons involving genes that represented independent phylogenetic lineages yielded very different divergence/conservation patterns. The distribution of stop codons in the F protein across all HCV genotypes was also investigated; genotypes 2 and 3 were found to have more stop codons than genotype 1. The results of this work suggest strongly that the pattern of divergence in the F protein is not affected by functional constraints.

Amino Acid Substitution↗
PubMed2001

Antioxidant enzyme expression in clinical isolates of Pseudomonas aeruginosa: identification of an atypical form of manganese superoxide dismutase.

Expression of superoxide dismutases (FeSOD and MnSOD) and catalases by laboratory strains of Pseudomonas aeruginosa is modulated by exogenous factors. Whether clinical isolates behave similarly and whether antioxidant enzyme expression influences P. aeruginosa virulence remain unclear. Fifty-seven P. aeruginosa blood culture isolates, plus seven pairs of blood and local-site isolates, were examined for FeSOD, MnSOD, and catalase production in vitro. Under iron-replete growth conditions FeSOD and catalase activities were maximized. MnSOD was not detected. FeSOD and catalase activity decreased under iron-limited growth conditions, whereas MnSOD activity appeared. SOD and catalase activity did not change with site of isolation or by patient. MnSOD could not be expressed by one isolate due to a missense mutation in sodA that produced a premature stop codon. Eleven percent of the isolates expressed a novel, rapidly migrating MnSOD that was associated with missense mutations in the normal stop codon of sodA. We conclude that clinical P. aeruginosa isolates vary little in FeSOD and catalase expression. Some strains produce a newly described MnSOD variant, whereas one is deficient in MnSOD production. The absence of MnSOD expression in a P. aeruginosa strain causing invasive human disease indicates that MnSOD is probably not essential for P. aeruginosa virulence.

Antioxidants↗
PubMed2003

Three novel mutations in SQSTM1 identified in familial Paget's disease of bone.

UNLABELLED: Mutations in Sequestosome 1 (SQSTM1) have been shown to segregate with familial Paget's disease of bone (PDB). We examined the coding sequence of SQSTM1 in five PDB pedigrees and found three novel mutations clustered around the C-terminal ubiquitin associated domain. Disruptions of the C-terminal domain of SQSTM1 seem to be a leading cause of familial PDB. INTRODUCTION: The characteristic features of Paget's disease of bone (PDB) are caused by focal areas of excessive and uncoordinated bone remodeling. A total of seven genetic loci (PDB1-PDB7) have been reported to be associated with the disease. The gene for Sequestosome 1 (p62; SQSTM1) has been identified as the causative gene for PDB3 in numerous French-Canadian families and families predominantly of British descent. To date, a total of three mutations, all affecting the ubiquitin-associated domain of SQSTM1, have been identified: a single 1215 C to T (P392L) transversion in exon 8, a T insertion in exon 8 (E396X), and a G to A mutation at the splice junction of exon 7 (IVS7 + 1). MATERIALS AND METHODS: DNA was isolated from blood collected from the members of five U.S. PDB pedigrees. Mutation analysis of the coding sequence of the SQSTM1 gene was performed on the proband and other key individuals in the pedigrees. RESULTS: Four of the five families had SQSTM1 mutations. Three of these mutations were novel: a single base deletion in exon 8 at position 1210 (1210delT) resulting in a premature stop codon at amino acid 394, a single C deletion in exon 8 at position 1215 (1215delC) also resulting in a premature stop codon at amino acid 394, and a single 1200 C to T (P387L) transversion in exon 7. CONCLUSION: Noteworthy is the fact that these three SQSTM1 mutations, in addition to the three previously described mutations, are clustered near the C-terminal of the protein. These mutations may be acting in a dominant-negative fashion to disrupt the ubiquitin-binding function, which could result in abnormal activation of the NF-kappaB pathway and the subsequent activation of the osteoclasts. These findings imply that SQSTM1 mutations may play a role in the majority of familial PDB in the United States.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Human lipoprotein lipase last exon is not translated, in contrast to lower vertebrates.

We have sequenced the first fish (zebrafish, Brachydanio rerio) lipoprotein lipase (LPL) cDNA clone. Similarities were found in mammalian LPL cDNA, but the codon spanning the last two exons (which is thus split by the last intron) is AGA (Arg) as opposed to TGA in mammals. Exon 10 is thus partially translated. These results were confirmed with rainbow trout (Oncorhynchus mykiss). We also investigated whether mammal TGA coded for selenocystein (SeCys), the 21st amino acid, but found that this was not the case: TGA does not encode SeCys but is a stop codon. It thus appears that the sense codon AGA (fish) has been transformed into a stop codon TGA (human) during the course of evolution. It remains to be determined if the "loss" of the C-terminal end of mammalian LPL protein has conferred an advantage in terms of LPL activity or, on the contrary, a disadvantage (e.g., susceptibility to diabetes or atherosclerosis).

Animals↗
PubMed2005

Mono-allelic POLG expression resulting from nonsense-mediated decay and alternative splicing in a patient with Alpers syndrome.

Alpers syndrome is an autosomal recessive mitochondrial DNA depletion disorder that affects children and young adults. It is characterized by a progressive, fatal brain and liver disease. This syndrome has been associated with mutations in POLG, the gene encoding the mitochondrial DNA polymerase (pol gamma). Most patients with Alpers syndrome have been found to be compound heterozygotes, carrying two pathogenic mutations in trans at the POLG locus. POLG is a nuclear-encoded gene whose protein product is imported into mitochondria, where it is essential for mtDNA replication and repair. We studied the skin fibroblasts of a patient with Alpers syndrome having the genotype E873stop/A467T. The E873stop mutation produces a premature termination codon (TAG) in exon 17. The A467T mutation produces a threonine to alanine substitution at a highly conserved site in exon 7. The allele bearing the stop codon (E873-TAG) is predicted to produce a truncated, catalytically inactive polymerase. However, only full-length pol gamma protein was detected by Western blot analysis. Here, we show that transcripts containing this stop codon undergo nonsense-associated alternative splicing and nonsense-mediated decay. More than 95% of the functional POLG mRNA was derived from the allele bearing the A467T mutation and less than 5% contained the E873stop mutation. These events ensured that virtually all POLG protein in the cell was expressed from the A467T allele. Therefore, the Alpers phenotype in this patient was a consequence of a single-copy gene dose of the A467T allele, and selective elimination of transcripts bearing the E873stop mutation.

Alleles↗
PubMed1994

The protein p30, encoded at the gag-pro junction of mouse mammary tumor virus, is a dUTPase fused with a nucleocapsid protein.

A ribosomal frameshift at the gag-pro junction of mouse mammary tumor virus (MMTV) gives rise to the protein p30. The protein consists of two domains, the zinc-finger-containing nucleocapsid (NC) protein portion with 95 residues and a C-terminal extension comprising 154 residues. The C-terminal domain shows similarity in sequence with the enzyme dUTPase from other sources. In this paper, we demonstrate that p30 is a functional dUTPase. Overproduction of the NC protein in Escherichia coli, using the native frameshift sequence at the gag stop codon, caused a detectable expression of dUTPase ascribed to a low frequency of readthrough. By a 1-base insertion, eliminating the gag stop codon and fusing the gag and pro reading frames, a plasmid, pET-3d-NCDU, directing overexpression of p30, was constructed. The overproduced protein, purified by phosphocellulose chromatography, shows both zinc-binding and dUTPase activity. Analytical gel filtration and sequence homology to other dUTPases suggest a trimeric assembly of p30 subunits. MMTV thus possesses two different forms of the nucleocapsid protein, the ordinary NC protein and the p30, having the NC protein connected to a domain of dUTPase.

Amino Acid Sequence↗
PubMed1989

Genetic structure of the bacteriophage P22 PL operon.

The sequence of 1416 base-pairs of the P22 PL operon was determined, linking a continuous sequence from PL through abc2. P22 mutants bearing deletions in the sequenced region were constructed and tested for their phenotypes. Plasmids were constructed to express PL operon genes singly and in combination from Plac UV5. Two previously known genes, 17 and c3, are located within this sequence. In addition, three new genes have been identified: ral, kil and arf. Genes ral and c3 are homologous, as well as functionally analogous, to lambda ral and cIII, respectively. P22 kil, like lambda kil, kills the host cell when it is expressed. The two kil genes, although analogous in cell killing and map location, have no apparent sequence homology. The functions of the P22 and lambda kil genes are unknown; however, P22 kil is essential for lytic growth in the absence of abc. Gene arf (accessory recombination function) is located just upstream from erf; it is essential for P22 growth in the absence of kil or other genes upstream in PL. The growth defect of P22 bearing a deletion that removes arf is complemented by expression of either arf or the lambda red genes from plasmids. Sequences that include the stop codon for gene 17 may form a small stem-loop structure and are nearly identical to lambda sequences that contain the stop codon for ssb, which is near lambda tL 2b. Plasmids that include the P22 structure negatively regulate kil gene expression in cis.

Amino Acid Sequence↗
PubMed2006

A frameshift in the coding region of a novel tomato class I basic chitinase gene makes it a pseudogene with a functional wound-responsive promoter.

A putative class I basic chitinase gene, assigned as psiBCH, was cloned from a tomato breeding line NC 24E. The gene contains a coding region with two introns. The predicted psiBCH open reading frame (ORF) is 971 bp and exhibits 81-88% identity at the nucleotide level with known class I basic chitinase genes from the Solanaceae family. However, the presence of a stop codon caused by a frameshift in the ORF of psiBCH makes it unusual among the other class I plant basic chitinases. This stop codon might be involved in the lower accumulation of fully spliced psiBCH RNA caused by nonsense-mediated decay (NMD), which is an RNA surveillance system universally found in eukaryotes. Sequence analysis of the 1883-bp 5'-flanking region of the psiBCH gene revealed the presence of potential wound-response promoter elements. To study the transcriptional regulation of the psiBCH gene, its 5'-flanking region containing the putative promoter was fused to the gus reporter gene and introduced into the tobacco genome via Agrobacterium tumefaciens-mediated transformation. Transgenic plants were functionally assayed for beta-glucuronidase activity. The psiBCH promoter drives the reporter gene expression in response to wounding stimuli. psiBCH promoter-GUS analysis indicates that wound-response of the tobacco transgene was rapid and localized in the wounded area following mechanical wounding. Therefore, our results suggest that the psiBCH promoter can provide targeted expression of genes, such as protease inhibitors in response to pest attack.

Agrobacterium tumefaciens↗
PubMed2006

Interactions of the release factor RF1 with the ribosome as revealed by cryo-EM.

In eubacteria, termination of translation is signaled by any one of the stop codons UAA, UAG, and UGA moving into the ribosomal A site. Two release factors, RF1 and RF2, recognize and bind to the stop codons with different affinities and trigger the hydrolysis of the ester bond that links the polypeptide with the P-site tRNA. Cryo-electron microscopy (cryo-EM) results obtained in this study show that ribosome-bound RF1 is in an open conformation, unlike the closed conformation observed in the crystal structure of the free factor, allowing its simultaneous access to both the decoding center and the peptidyl-transferase center. These results are similar to those obtained for RF2, but there is an important difference in how the factors bind to protein L11, which forms part of the GTPase-associated center of the large ribosomal subunit. The difference in the binding position, C-terminal domain for RF2 versus N-terminal domain for RF1, explains a body of L11 mutation studies that revealed differential effects on the activity of the two factors. Very recent data obtained with small-angle X-ray scattering now reveal that the solution structure of RF1 is open, as here seen on the ribosome by cryo-EM, and not closed, as seen in the crystal.

Cryoelectron Microscopy↗
PubMed2003

Long-patch DNA repair synthesis during base excision repair in mammalian cells.

The base excision repair (BER) process removes base damage such as oxidation, alkylation or abasic sites. Two BER sub-pathways have been characterized using in vitro methods, and have been classified according to the length of the repair patch as either 'short-patch' BER (one nucleotide) or 'long-patch' BER (LP-BER; more than one nucleotide). To investigate the occurrence of LP-BER in vivo, we developed an assay using a plasmid containing a single modified base in the transcribed strand of the enhanced green fluorescent protein (EGFP) gene and a stop codon, based on a single-nucleotide mismatch, at varying distances on the 3' side of the lesion. The reversion of the stop codon occurs after DNA repair synthesis and restores EGFP expression after transfection of mismatch-repair-deficient cells. Repair patches longer than one nucleotide were observed for 55-80% or 80-100% of the plasmids with a mean length of 2-6 or 6-12 nucleotides for 8-oxo-7,8-dihydroguanine or a synthetic abasic site, respectively. These data show the existence of LP-BER in vivo, and emphasize the effect of the type of BER substrate lesion on both the yield and the extent of the LP-BER sub-pathway.

Animals↗
PubMed1996

A molecular analysis of viral persistence in surface antigen-negative chronic hepatitis B.

To identify the mechanisms of viral persistence in patients with chronic hepatitis B after the acquisition of anti-hepatitis B surface antigen antibodies (antiHBs), we serially analyzed the nucleotide sequence of the envelope region in a cohort of infected patients. Four patients with histological diagnoses of chronic hepatitis B who had at least 5 years of observance by our hospital staff were studied. All but one showed normalization of serum alanine aminotransferase (ALT) concentration after clearance of the hepatitis B surface of antigen (HBsAg) and the appearance of anti-HBs. Hepatitis B virus (HBV) DNA was still detectable by polymerase chain reaction (PCR) amplification assay in serum specimens from two patients, even in the presence of circulating anti-HBs. The envelope gene was amplified by PCR in serum samples obtained both before and after seroconversion, and direct cycle sequencing of the PCR products was performed. A mutation resulting in a premature stop codon was found in the pre-S1 region of one patient just prior to clearance of HBsAg. Two years later, the stop codon was converted to a leucine codon and three mutations developed in the "a" loop. In the other patient, 16 amino acids had been deleted between amino acids 8 and 23 in the pre-S2 region before clearance of HBsAg. After the appearance of circulating anti-HBs, the pre-S2 gene reverted to the wild type but three additional mutations appeared inside the "a" loop. These results suggest that HBV mutates when HBsAg is cleared, which may contribute to viral persistence due to an evasion of the host immune surveillance.

Amino Acid Sequence↗
PubMed1993

Splice variants of the N-methyl-D-aspartate receptor NR1 identify domains involved in regulation by polyamines and protein kinase C.

The N-methyl-D-aspartate (NMDA) receptor NR1 gene encodes RNA that is alternatively spliced to generate at least seven variants. The variants arise from splicing in or out of three exons; one encodes a 21-amino acid insert in the N-terminal domain, and two encode adjacent sequences of 37 and 38 amino acids in the C-terminal domain. Splicing out of the second C-terminal exon deletes a stop codon and results in an additional open reading frame encoding an unrelated sequence of 22 amino acids before arriving at a second stop codon. We denote the NR1 variants by the presence or absence of the three alternatively spliced exons (from 5' to 3'); thus, NR1(111) has all three exons, NR1(000) has none, and NR1(100) has only the N-terminal exon. We report here electrophysiological characterization of six splice variants of the NR1 receptor expressed in Xenopus oocytes. NR1 receptors that lacked the N-terminal exon (NR1(000), NR1(010), and NR1(011)) exhibited a relatively high affinity for NMDA (EC50 approximately 13 microM) and marked potentiation by spermine. In contrast, those receptor variants with the N-terminal insert (NR1(100), NR1(101), and NR1(111)) showed a lower agonist affinity and little or no spermine potentiation at saturating glycine. All six variants showed spermine potentiation at low glycine and inhibition by spermine at more negative potentials. Variants differing only in the C-terminal domain differed little in agonist affinity and spermine potentiation. These findings indicate that the N-terminal insert either participates in agonist and polyamine binding domains or indirectly modifies their conformations. The splice variants differed in the extent to which they could be potentiated by activators of protein kinase C (PKC) from 3- to 20-fold. Presence of the N-terminal insert and absence of the C-terminal sequences increased potentiation by PKC. These findings identify the contributions of the separate polypeptide domains to modulation by polyamines and PKC and provide further support for the concept that subunit composition determines functional properties of NMDA receptors.

Alternative Splicing↗
PubMed2004

Truncation of fibronectin-binding proteins in Staphylococcus aureus strain Newman leads to deficient adherence and host cell invasion due to loss of the cell wall anchor function.

Staphylococcus aureus fibronectin-binding proteins (FnBPs) play a critical role in S. aureus pathogenesis. FnBPs mediate adhesion to fibronectin and invasion of mammalian cells, including epithelial, endothelial, and fibroblastic cells, by fibronectin bridging to the host cell fibronectin receptor integrin (alpha(5))beta(1). Strain Newman is a laboratory strain frequently used for genetic, functional, and in vivo studies. However, despite pronounced production of FnBPs, strain Newman is only weakly adherent to immobilized Fn and weakly invasive. We examined whether these effects are due to a structural difference of FnBPs. Here, we show that both fnbA(Newman) and fnbB(Newman) contain a centrally located point mutation resulting in a stop codon. This leads to a truncation of both FnBPs at the end of the C domain at identical positions. Most likely, the stop codon occurred first in fnbB(Newman) and was subsequently transferred to fnbA(Newman) by replacement of the entire region encompassing the C, D, and W domains with the respective sequence of fnbB(Newman). Using heterologous expression in Staphylococcus carnosus, we found that truncated FnBPs were completely secreted into the culture medium and not anchored to the cell wall, since they lack the sortase motif (LPETG). Consequently, this led to a loss of FnBP-dependent functions, such as strong adhesion to immobilized fibronectin, binding of fibrinogen, and host cell invasion. This mutation may explain some of the earlier reported conflicting data with strain Newman. Thus, care should be taken when drawing negative conclusions about the role of FnBPs as a virulence factor in a given model.

Adhesins, Bacterial↗
PubMed2004

Genetic complementation reveals a novel human congenital disorder of glycosylation of type II, due to inactivation of the Golgi CMP-sialic acid transporter.

We have identified a homozygous G>A substitution in the donor splice site of intron 6 (IVS6 + 1G>A) of the cytidine monophosphate (CMP)-sialic acid transporter gene of Lec2 cells as the mutation responsible for their asialo phenotype. These cells were used in complementation studies to test the activity of the 2 CMP-sialic acid transporter cDNA alleles of a patient devoid of sialyl-Le(x) expression on polymorphonuclear cells. No complementation was obtained with either of the 2 patient alleles, whereas full restoration of the sialylated phenotype was obtained in the Lec2 cells transfected with the corresponding human wild-type transcript. The inactivation of one patient allele by a double microdeletion inducing a premature stop codon at position 327 and a splice mutation of the other allele inducing a 130-base pair (bp) deletion and a premature stop codon at position 684 are proposed to be the causal defects of this disease. A 4-base insertion in intron 6 was found in the mother and is proposed to be responsible for the splice mutation. We conclude that this defect is a new type of congenital disorder of glycosylation (CDG) of type IIf affecting the transport of CMP-sialic acid into the Golgi apparatus.

Alternative Splicing↗
PubMed2004

Sheep exhibit novel variations in the organization of the mammalian type II gonadotropin-releasing hormone receptor gene.

Species-specific differences in genes encoding type II GnRH receptor indicate that a functional hepta-helical receptor is produced in monkeys but not in rodents, cows, chimpanzees, or humans. To further investigate the extent of evolutionary differences, we sequenced the type II GnRH receptor gene from wild-type Soay sheep. The gene was isolated by long-distance PCR using primers to PEX11beta and RBM8A genes known to flank type II GnRH receptor gene homologues. The gene spans 5.7-kb DNA and was sequenced after shot-gun subcloning. Its novel features include absence of a Pit-1 transcription factor binding site, a premature stop codon (TAG) in exon 1, an in-frame deletion of 51 bp (17 codons) in exon 2, and several nonconservative codon changes. Sheep breed variation in the gene was assessed using genomic DNA in PCR-restriction digest assays for the premature stop codon and in a PCR assay for the deletion. Both characteristics were present in all 15 breeds tested. Receptor gene expression was investigated using poly-A(+) RNA Northern analysis, RT-PCR, and in situ hybridization. An oligonucleotide probe to exon 1 revealed an alternative transcript in testis but not in pituitary gland. No transcripts in testis or pituitary were detectable using an exon 2-3 probe. All tissues examined including multiple brain areas and gonadotrope-enriched cell cultures were negative for type II GnRH receptor in RT-PCR. Testis and pituitary sections were negative with exon 1 riboprobes and exon 1 or 2-3 oligonucleotide probes in in situ hybridization. A hepta-helical type II GnRH receptor is therefore not expressed from this sheep gene.

Alternative Splicing↗