Cloning and nucleotide sequence of the equine and elk pituitary pre-prolactin cDNA

We report the equine (Equs equs) and elk (Cervus elaphus) pituitary pre-prolactin (PRL) cDNA cloning, and their nucleotide and deduced amino acid sequences. Pre-PRL cDNA was obtained by RNA ligation mediated-rapid amplification of cDNA ends (RLM-RACE) and polymerase chain reaction (PCR). The elk pre-PRL cDNA exhibits two polymorphisms at positions 96 and 672, which are silent since they encode for the same amino acids, proline and isoleucine, respectively. We found no polymorphisms in the equine pre-PRL cDNA. The deduced amino acid sequence of the equine pre-PRL is 99% identical to the previously reported protein sequence. Pre-PRL mRNA is <1 kb in length and is highly expressed in the anterior pituitary gland, as demonstrated by Northern hybridization analysis. In summary, we cloned and sequenced the equine and elk pre-PRL cDNAs. The deduced amino acid sequence of elk and equine pre-PRL appears to be moderately conserved among other mammalian species. The polymorphic sites found in the elk cDNA could potentially be used in parentage testing and gene mapping.     

Nucleotide and deduced amino acid sequence of equine retinal and pineal gland phosducin

OBJECTIVES: To determine the full-length complementary DNA (cDNA) sequence of equine retinal and pineal gland phosducin (PHD) and to clone these sequences. SAMPLE POPULATION: Samples of equine retinal RNA. PROCEDURE: A primer set was designed for use in identifying a fragment of the equine PHD nucleotide sequence, derived from retinal RNA samples, and subsequently for use to deduce specific primers for additional examination. The full-length cDNA was determined by the method of rapid amplification of cDNA ends (RACE). For full-length cDNA, newly designed primers were used. Nucleotide sequences were analyzed by use of computer software. The deduced amino acid sequence was compared with sequences of PHD reported for other species. In addition, the sequence of equine pineal PHD was cloned. RESULTS: The cDNA nucleotide sequence for equine PHD was 1,209 base pairs (bp) in length with an open-reading frame encoding a protein of 245 amino acids and a calculated molecular mass of 28.214 kd. Similarity with amino acid sequences of PHD from other species was 89 to 93%. Sequences of equine PHD from retina and pineal gland were identical. Equine PHD contained a peptide sequence with 100% homology to an uveitopathogenic peptide reported for rat PHD. CONCLUSIONS: Equine PHD is a highly conserved protein that has homology of immunologic interest with rat PHD. These results establish a basis for studying the role of PHD in ocular inflammation of horses.     

A light and electron microscopic study of a normal adrenal medulla and a pheochromocytoma from a horse.

The outer medullary (juxtacortical) zone of a normal equine adrenal gland had columnar chromaffin-positive cells arranged with their long axes perpendicular to fine vascular channels. The deeper medullary regions were composed of smaller irregularly round to polygonal chromaffin positive cells in small packets. Both cell types contained two types of membrane-bound cytoplasmic secretory granules. Osmiophilic granules with a homogeneous core, crenated membrane and narrow submembranous halo predominated in the columnar juxtacortical cells. The rounder, central medullary cells contained predominantly electron dense granules with a wide irregular electron lucent space between an eccentric core and the granule membrane. In contrast, irrespective of cell type or zone, cells from a pheochromocytoma contained only one type of granule similar to that described for the juxtacortical region of the normal equine adrenal medulla. The tumor cells could be classified into three subtypes based on density of granule packing but the granules were morphologically similar in all tumor cells.     

Molecular cloning and sequencing of equine cDNA encoding serum amyloid A (SAA)

The serum amyloid A (SAA) protein is a characteristic and sensitive acute phase reactant in all vertebrates investigated. We molecularly cloned the equine cDNA encoding SAA from the liver of a healthy horse by polymerase chain reaction (PCR). The cloned cDNA is 480 bases in length, and contains an open reading frame (ORF) of 387 nucleotides encoding a precursor SAA protein of 128 amino acids. The precursor of horse SAA seems to have an 18-residue signal peptide and differs from the reported amino acid sequences of the horse SAA by substitution of valine at residue 81. It shows high homology with SAA amino acid sequence of other species such as dog (80.6%), mink (77.5%), human (76.9%) and duck (71.9%). An insertion of eight amino acids at residues between 85 and 92, as compared to human SAA, has also been found in horse SAA. The availability of the equine SAA cDNA will provide a useful reagent for studying its role in diseased horses.     

Molecular cloning, sequencing, and expression of equine interleukin-6

Equine interleukin-6 (IL-6) cDNA was amplified from mitogen-stimulated equine peripheral blood mononuclear cells (PBMC) using consensus sequence primers. The 727bp amplified cDNA contains the entire coding region for equine IL-6 and includes 118 bases in the 3' non-translated region. The coding sequence translates to a protein of 208 amino acids with a predicted 28 amino acid leader sequence. The mature protein of 180 amino acids has a predicted molecular mass of 20471Da without post-translational modifications. The amino acid sequence of equine IL-6 displays between 46 and 84% similarity to other mammalian IL-6 sequences. Expression of equine IL-6 in Chinese hamster ovary (CHO) cells yielded a supernatant that supported the proliferation of B9 cells in a dose-dependent manner. Treatment of B9 cells with an anti-IL-6 receptor antibody ablated the response to the recombinant equine IL-6.     

Cloning of canine Toll-like receptor 7 gene and its expression in dog tissues

Toll-like receptor 7 (TLR7) is activated by single strand RNA and imidazoquinoline compounds, and induces interferon production. In this study, canine TLR7 cDNA was cloned and sequenced. The full-length cDNA of canine TLR7 gene was 3419bp, encoding 1032 amino acids. The similarities of canine TLR7 with human and mouse TLR7 were 84 and 80% at the nucleotide sequence level, and 86 and 79% at amino acid sequence level, respectively. Further, the expression of TLR7 mRNA was investigated in canine normal tissues by semiquantitative RT-PCR analysis. The common expression level of TLR7 mRNA in tissues from three dogs examined was in large intestine, lung, pancreas, small intestine and skin, though the expression level in each tissue was varied among these healthy dogs. In other tissues (kidney, liver, lymph node, spleen, adrenal gland, and PBMCs), the level of TLR7 mRNA expression was different in individuals.     

Molecular characterization and functional expression of equine interleukin-1 type I and type II receptor cDNAs

cDNA generated from lipopolysaccharide-stimulated equine peripheral blood mononuclear cells was used to amplify and clone type I and type II equine interleukin-1 receptors (IL-1RI and IL-1RII) using primers derived from semi-conserved regions between human and mouse IL-1RI and IL-1RII sequences, respectively. 5' and 3' terminal sequences of equine IL-1RI and IL-1RII were amplified by 5' and 3' rapid amplification of cDNA ends. The deduced amino acid sequence of equine IL-1RI demonstrated 77, 64 and 63% similarity with human, mouse and rat sequences, respectively. The predicted amino acid sequence of equine IL-1RII demonstrated 70, 60 and 58% similarity with human, mouse and rat sequences, respectively. Recombinant equine soluble IL-1RI and IL-1RII produced in insect cells bound recombinant equine IL-1alpha and IL-1beta. Furthermore, both receptors suppressed the growth inhibitory activities of equine IL-1alpha and IL-1beta toward A375 cells in a dose-dependent manner, indicating that the present equine IL-1RI and IL-1RII cDNA encodes biologically active proteins.     

西农萨能羊两个泌乳阶段差异表达基因SAA3的克隆和序列分析

为了研究不同泌乳期乳腺组织中差异表达基因，利用抑制性削减杂交技术构建西农萨能羊泌乳60d乳腺组织及泌乳28d乳腺组织中差异表达基因文库，用Real-Time PCR技术验证阳性克隆血清淀粉样蛋白A3（SAA3）基因，通过RT-PCR方法克隆西农萨能羊乳腺组织SAA3，并进行序列比对和功能预测。结果成功构建了西农萨能羊不同泌乳期乳腺组织中差异表达基因文库，筛选克隆了乳腺组织SAA3基因，GenBank登录号为：DQ839400，编码区长度为396bp，含有131个氨基酸。西农萨能羊乳腺组织中SAA3与牛（GenBank：NM_181016）、兔（GenBank：M64696．1）、人（GenBank：BC020795）、鼠（GenBank：NM_011315）核苷酸同源性分别为95％、84．3％、81.3％和81.9％，氨基酸同源性为93％、76％、72％、72％；在编码区261-287位较人、兔、鼠SAA编码区多27个碱基，连续大于5个氨基酸的保守区域有6个，较人SAA多3个潜在功能基序。     

猪伪狂犬病病毒受体nectin-2基因的克隆与分子特征

为了进一步了解猪nectin-2基因的结构与功能,本研究采用生物信息学结合RT-PCR的方法从猪脑组织中克隆到了猪nectin-2基因,并对其核苷酸序列和推导的氨基酸序列进行了比较分析。猪nectin-2基因的编码区含有1440个核苷酸,编码479个氨基酸,其中信号肽由32个氨基酸组成,胞外域由330个氨基酸组成,含有2个潜在的N-糖基化位点和6个半胱氨酸残基,跨膜区由23个氨基酸组成,胞浆区由94个氨基酸组成,猪nectin-2基因与犬、马、家鼠、人、恒河猴、牛、黑猩猩的nectin-2基因核苷酸序列同源性分别为85.4%、85.7%、78.6%、82.1%、82.1%、81.9%和82.1%;推导氨基酸序列的同源性分别为84.5%、83.0%、74.7%、75.7%、76.4%、78.4%和75.5%。本试验为进一步深入研究猪伪狂犬病病毒与宿主之间的关系奠定了基础。     

Expressed gene sequence of the IFNγ-response chemokine CXCL9 of cattle, horses, and swine

This report describes the cloning and characterization of expressed gene sequences of bovine, equine, and swine CXCL9 from RNA obtained from peripheral blood mononuclear cells (PBMC) and other tissues. The bovine coding region was 378 nucleotides in length, while the equine and swine coding regions were 381 nucleotides. Mapping showed that all three sequences were coded for in four exons in the genome, as are the human and mouse genes. The bovine, equine, and swine coding regions shared 83%, 86%, and 84% homology with human CXCL9, respectively, and all three were 74% homologous with mouse CXCL9. Cladogram comparison of the nucleotide sequences of CXCL9 showed that the bovine, equine and swine sequences were more closely related to one another than to either the human or the mouse sequences. However, the human sequence was more closely related to them than it was to the mouse sequence. These relationships were preserved when the deduced amino acid sequences were evaluated and all sequences showed conservation of the characteristic four cysteines. This work sets the stage for further work with these molecules; an integral goal of the U.S. Veterinary Immune Reagent Network is to develop reagents for investigating diseases in livestock species, poultry, and fish.     

Analysis of the matrix protein gene sequence of the Asian lineage of peste-des-petits ruminants vaccine virus

The M gene nucleotide sequence of an Indian peste-des-petits ruminants (PPRV) vaccine virus ("PPRV Sungri/96") belonging to Asian lineage was determined. The gene is 1476 nucleotides long with a single open reading frame (ORF). The nucleotide and predicted amino acid sequence was compared with the homologous region of the African Lineage Vaccine virus "PPRV/Nigeria/75/1". The nucleotide sequence of the "PPRV Sungri/96" was 86% identical to that of "PPRV/Nigeria/75/1", while a homology of 93% and 95% could be observed in the ORF and amino acids level, respectively. The M gene encodes a protein of 335 amino acids, with a predicted molecular weight (MW) of 37.8 kDa. The ORF is flanked by a 3' untranslated region of 436 nucleotides and a high level of sequence divergence (approximately 30%) could be observed in this region between the vaccine viruses of Asian and African lineages. A high degree of conservation of several amino acids of this protein observed previously was also confirmed in this study.     

Nucleotide sequence and genetic analysis of the leader region of Canadian, American and European equine arteritis virus isolates.

The extreme 5' end, the entire leader sequence of the Arvac vaccine strain, and 10 equine arteritis virus (EAV) isolates, including the ATCC Bucyrus reference strain and 5 Canadian field isolates, were determined and compared at the primary nucleotide and secondary structure levels. The leader sequence of eight EAV isolates, including the Bucyrus reference strain, and the leader sequence of the Arvac vaccine strain was determined to be 206 nt in length (not including the putative 5' cap structure-associated nucleotide) whereas those of the 86AB-A1 and 86NY-A1 isolates were found to be 205 and 207 nt in length, respectively. The sequence identity of the leader sequences, between the different isolates and the Bucyrus reference strain, ranged from 94.2 to 98.5%. Phylogenetic analysis and estimation of genetic distances, based on the leader nucleic acid sequences, showed that all EAV isolates/strains are likely to represent a large phylogenetically-related group. An AUG start codon found at position 14 in all EAV isolates/strains could initiate an open reading frame (ORF) that could produce a polypeptide of 37 amino acids, except for the 86NY-A1 isolate where the intraleader polypeptide would contain 54 amino acids. Computer-predicted RNA secondary structures were identified in the 11 EAV leader regions analyzed. All EAV isolates/strains showed 3 conserved stem-loops (designated A, B and C). An additional conserved stem-loop (D) was observed in 7 EAV isolates, including the Bucyrus reference strain. The leader region distal to stem-loop D did not contain conserved sequences or stem-loop structures common to the EAV isolates/strains.     

Phylogenetic and structural studies of a novel equine papillomavirus identified from aural plaques

Papillomaviruses (PVs) infect a wide range of animal species and show great genetic diversity. To date, excluding equine sarcoids, only three species of PVs were identified associated with lesions in horses: Equus caballus papillomavirus 1 (EcPV1-cutaneous), EcPV2 (genital) and EcPV3 (aural plaques). In this study, we identified a novel equine PV from aural plaques, which we designated EcPV4. Cutaneous samples from horses with lesions that were microscopically diagnosed as aural plaques were subjected to DNA extraction, amplification and sequencing. Rolling circle amplification and inverse PCR with specific primers confirmed the presence of an approximately 8 kb circular genome. The full-length EcPV4 L1 major capsid protein sequence has 1488 nucleotides (495 amino acids). EcPV4 had a sequence identity of only 53.3%, 60.2% and 51.7% when compared with the published sequences for EcPV1, EcPV2 and EcPV3, respectively. A Bayesian phylogenetic analysis indicated that EcPV4 clusters with EcPV2, but not with EcPV1 and EcPV3. Using the current PV classification system that is based on the nucleotide sequence of L1, we could not define the genus of the newly identified virus. Therefore, a structural analysis of the L1 protein was carried out to aid in this classification because EcPV4 cause lesion similar to the lesion caused by EcPV3. A comparison of the superficial loops demonstrated a distinct amino acid conservation pattern between EcPV4/EcPV2 and EcPV4/EcPV3. These results demonstrate the presence of a new equine PV species and that structural studies could be useful in the classification of PVs.     

猪Toll样受体9基因的克隆、序列分析及其结构预测

本研究应用反转录-聚合酶链式反应(RT-PCR)扩增技术,从猪脾脏淋巴细胞中,克隆了猪Toll样受体9基因(pTLR9).基因序列分析表明,克隆的pTLR9基因ORF为3 093 bp,编码1 030个氨基酸,含18.5%的亮氨酸,含有24个氨基酸的信号肤序列,属于Ⅰ型跨膜受体,具有富含亮氨酸的重复序列(LRR)和Toll/IL-1R同源区结构域;与GenBank上登载的pTLR9参考序列(AY859728)的同源性为99.3%,与牛、马、羊和人的同源性较高,与家鼠、褐鼠的次之,TLR9的演化关系与亲缘关系密切.     

禽呼肠病毒P10、P17非结构蛋白基因的克隆及序列分析

根据GenBank上的禽呼肠病毒(ARV)S1基因序列,设计并合成了一对跨越P10和P17非结构蛋白基因的特异性引物,对13个ARV毒株进行RT-PCR扩增、克隆及序列测定。结果显示,13个ARV毒株的P10蛋白基因ORF全长均为297bp,编码98个氨基酸;P17蛋白基因ORF全长为441bp,编码146个氨基酸。这13个ARV毒株P10、P17蛋白基因核苷酸同源性分别在96.6%~100%和95.2%~99.3%之间,推导的氨基酸同源性分别在98.2%~100%和91.9%~99.0%之间。将这13个ARV毒株与GenBank上其他正呼肠病毒毒株,包括番鸭株(DRV)和飞狐上分离到的内尔森海湾病毒(NelsonBayvirus,NBV)及两个澳洲分离株(ARM-1和SOM-4)进行同源性比较和遗传进化树分析,结果表明,呼肠病毒有地域和种类的差别。     

Cloning and sequence analysis of the complementary DNA for feline preproparathyroid hormone

OBJECTIVES: To clone and sequence the cDNA for feline preproparathyroid hormone (preproPTH) and to compare that sequence with other known parathyroid hormone (PTH) sequences. SAMPLE POPULATION: Parathyroid glands from 1 healthy cat. PROCEDURES: A cDNA library was constructed in lambda phage from feline parathyroid gland mRNA and screened with a radiolabeled canine PTH probe. Positive clones were sequenced, and nucleic acid and deduced amino acid sequences were analyzed and compared with known preproPTH and PTH sequences. RESULTS: Screening of approximately 2 X 10(5) recombinant plaques revealed 3 that hybridized with the canine PTH probe; 2 clones comprised the complete sequence for feline preproPTH. Feline preproPTH cDNA consisted of a 63-base pair (bp) 5'-untranslated region (UTR), a 348-bp coding region, and a 326-bp 3'-UTR. The coding region encoded a 115-amino acid peptide. Mature feline PTH consisted of 84 amino acids. Amino acid sequence analysis revealed that feline PTH was > 83% identical to canine, bovine, swine, equine, human, and macaque PTH and 69, 71, and 44% identical to mouse, rat, and chicken PTH, respectively. Within the region responsible for hormonal activity (amino acids 1 to 34), feline PTH was > 79% identical to other mammalian PTH sequences and 64% identical to the chicken sequence. CONCLUSIONS AND CLINICAL RELEVANCE: The amino acid sequence of PTH is conserved among mammalian species. Knowledge of the cDNA sequence for feline PTH may be useful to investigate disturbances of calcium metabolism and alterations in PTH expression in cats.