Expression of the bovine oestrogen receptor-beta (bERbeta) messenger ribonucleic acid (mRNA) during the first ovarian follicular wave and lack of change in the expression of bERbeta mRNA of second wave follicles after LH infusion into cows

In a previous study, the ERbeta cDNA protein-coding region was utilised to clone bovine ERbeta. The objectives in this study were to examine (1) ERbeta mRNA expression in ovarian follicles throughout the bovine first follicular wave, and (2) effect of LH infusion into cows on bERbeta mRNA expression during the second follicular wave. In experiment 1, heifers (4-5 per time point) were ovariectomized at 12, 24, 36, 48, 60, 72, 84, 96, 144, or 216 h after emergence of the first follicular wave after oestrus. In experiment 2, saline or LH was pulsed hourly (computer-controlled syringe pump) into cows (n = 31; 5-6 per treatment) at wave emergence for 2 or 4 days: wave 1-saline (W1S), wave 2-saline (W2S), or wave 2-LH (25 microg/h; W2LH). Ovaries were removed on day 2 or day 4 after wave emergence. Follicles, 2-19mm in size, were dissected, frozen, and stored at -80 degrees C for in situ hybridisation with two bERbeta cRNA probes. Expression of bERbeta mRNA was localised in granulosa cells of healthy follicles. In experiment 1, bERbeta mRNA expression did not change with time points of the wave showing no association of bERbeta mRNA expression with follicular selection and dominance. However, bERbeta mRNA expression decreased with increase in size of all follicles. Expression of bERbeta mRNA was greater in very small follicles (2-4 mm) than in large (> or = 9 mm) follicles. In experiment 2, expression of bERbeta mRNA in follicles did not differ either between W1S and W2S or between W2S and W2LH. In summary, bERbeta mRNA expression decreased with increasing follicular size. However, neither stage of the wave (selection or dominance), nor pulsatile infusion of LH influenced bERbeta mRNA expression.     

牛卵巢PRSS35基因CDS序列分析

为测定牛卵巢丝氨酸蛋白酶35 (PRSS35)的CDS序列并进行生物信息学分析。试验根据NCBI上已公布牛PRSS35基因的mRNA序列设计特异性引物,使用RT-PCR技术扩增牛卵泡中PRSS35的CDS序列。结果显示,牛PRSS35基因CDS区序列全长为1 239 bp,共编码412个氨基酸,PRSS35与其他10个物种的同源序列相似性较高,且该蛋白具有一个长度为20个氨基酸的信号肽,具有11个O-糖基化位点和2个N-糖基化位点,以及3个磷酸化位点,并发现有一个典型的Tryp_Spc结构域,即胰蛋白酶样丝氨酸蛋白酶结构域。为进一步研究该基因及其编码蛋白在卵泡发育过程中所起的作用提供了一定的理论依据。     

Rat pineal gland phosducin: cDNA isolation, nucleotide sequence, and chromosomal assignment in the mouse

The pineal gland contains a soluble phosphoprotein, phosducin, which is homologous to that of retinal photoreceptors. Phosducin has been shown to bind the beta, gamma subunits of the retinal G-protein transducin. Retinal phosducin has been cloned and now we report a rat pineal cDNA encoding phosducin. A 1217-nucleotide cDNA was isolated from a rat pineal library by DNA-DNA hybridization with a polymerase chain reaction-amplified cDNA of bovine retina mRNA for phosducin. Northern blot analysis demonstrates that the mRNA for phosducin is approximately 1.3 kb in both rat pineal and rat retina. The translated mRNA from rat pineal encodes a protein with 246 amino acids, compared to the 245 amino acids of bovine retina phosducin. The predicted molecular weight of rat pineal phosducin is 28,201. Immunoblot analysis with affinity-purified antibodies against bovine retina phosducin identify a single immunoreactive protein of approximately 33 kDa in both rat retina and rat pineal. The amino acid sequence of rat pineal phosducin is homologous to that of bovine retina phosducin, revealing 89% identity and another 5.7% similarity. Both rat pineal and bovine retina phosducins are acidic proteins with pIs of 4.3 and 4.5, respectively. The translated protein lacks hydrophobic domains that would suggest an integral membrane protein. Rat pineal phosducin has a single consensus phosphorylation domain for protein kinase A that is nearly identical to that of retinal phosducin, which is phosphorylated by protein kinase A in situ. Rat phosducin also contains three potential phosphorylation domains for protein kinase C and nine for casein kinase II as well as a predicted site for N-glycosylation. The cDNA encoding phosducin was used to localize the gene within a linkage group to a large segment of mouse chromosome 1 in a conserved region with the long arm of human chromosome 1 with a panel of DNA samples from an interspecific cross. In keeping with a proposed role of retinal phosducin in down-regulation of the photo-transduction cascade, a modulatory role in signal transduction is proposed for pineal phosducin.     

Insulin-like growth factors I and II in fetal and adult bovine serum. Purification, primary structures, and immunological cross-reactivities

Insulin-like growth factors I and II (IGF-I and IGF-II) were prepared from fetal calf serum and adult bovine serum by gel filtration, immunoaffinity chromatography, chromatofocusing, and reverse-phase high pressure liquid chromatography, and their complete amino acid sequences determined. IGF-I and -II are found in both adult and fetal serum. The sequence of bovine IGF-I is found to be identical to that of human IGF-I, whereas 3 out of 67 amino acid residues are found to be different between bovine and human IGF-II. The differences are located in the C-peptide region of the molecule. Bovine IGF-II shows less than 10% immunological cross-reactivity with antisera against human and rat IGF-II, but is equipotent to human IGF-II in displacing human 125I-labeled IGF-II from human placental receptor. Bovine IGF-I was equipotent to human IGF-I in both radioimmunoassays and radioreceptor assays within the limits of the assay.     

The partial amino acid sequence of bovine cartilage proteoglycan, deduced from a cDNA clone, contains numerous Ser-Gly sequences arranged in homologous repeats.

We have determined the sequence of a partial cDNA clone encoding the C-terminal region of bovine cartilage aggregating proteoglycan core protein. The deduced amino acid sequence contains a cysteine-rich region which is homologous with chicken hepatic lectin. This lectin-homologous region has previously been identified in rat and chicken cartilage proteoglycan. The bovine sequence presented here is highly homologous with the rat and chicken amino acid sequences in this apparently globular region. A region containing clusters of Ser-Gly sequences is located N-terminal to the lectin homology domain. These Ser-Gly-rich segments are arranged in tandemly repeated, approx. 100-residue-long, homology domains. Each homology domain consists of an approx. 75-residue-long Ser-Gly-rich region separated by an approx. 25-residue-long segment lacking Ser-Gly dipeptides. These dipeptides are arranged in 10-residue-long segments in the 100-residue-long homology domains. The shorter homologous segments are tandemly repeated some six times in each 100-residue-long homology domain. Serine residues in these repeats are potential attachment sites for chondroitin sulphate chains.     

Nucleotide and deduced amino acid sequence of bovine adrenal medulla chromogranin B (secretogranin I)

1. A novel 1745-dalton pyroglutamyl peptide (BAM-1745)6 was recently isolated and characterized from bovine adrenal medulla chromaffin granules. Its amino acid sequence was found to be 93% identical to residues 580-593 of human chromogranin B (secretogranin I). 2. Based on this sequence a degenerate oligonucleotide probe was synthesized and used to identify a 2.4-kb bovine adrenal medulla chromogranin B cDNA. 3. The deduced polypeptide is 647 amino acids long and begins with a putative signal sequence of 20 residues as in the human, rat, and mouse proteins. Also conserved in the bovine protein is a tyrosine residue which may be sulfated, two N-terminal cysteines, and many paired basic amino acids which may serve as sites of posttranslational processing. The peptide BAM-1745 is flanked by paired basic amino acids and therefore is most likely a product of posttranslational processing. Bovine chromogranin B is 67, 58, and 58% identical to the human, rat, and mouse chromogranin B proteins, respectively. 4. The carboxyl terminus of bovine chromogranin B, including BAM-1745, was found to be the most conserved region of the polypeptide and may identify it as an important functional domain.     

Molecular cloning, cDNA structure, and regulation of the regulatory subunit of type II cAMP-dependent protein kinase from rat ovarian granulosa cells

One isoform of the regulatory subunit of type II cAMP-dependent protein kinase (R-II51; Mr = 51,000) and its electrophoretic variants (R-II51.5 and R-II52; Mr = 51,500 and 52,000, respectively) are selectively induced by estradiol and follicle-stimulating hormone (cAMP) in rat ovarian granulosa cells. To ascertain the amino acid sequence of R-II51 and to gain insight into the molecular events regulating the intracellular content of ovarian follicular R-II51, we constructed a lambda gt11 cDNA expression library from poly(A)+ RNA of hormone-primed rat granulosa cells. A 1.5-kilobase (kb) cDNA insert, isolated from a plaque-purified R-II antibody positive bacteriophage clone, selectively bound R-II51 mRNA as demonstrated by analysis of the hybrid-selected translation product. Restriction maps and sequence analyses of the 1.5-kb cDNA insert and of the 1.8- and 2.2-kb cDNA inserts from two additional clones showed overlapping sequences which span a region of 3065 nucleotides in size. The 1.5- and 1.8-kb cDNA inserts each contained poly(A) addition signals (1508 and 1761 nucleotides, respectively), terminal poly(A) sequences, and the entire coding region for R-II51 (1204 nucleotides) except for a small number of nucleotides at the 5' end. The 2.2-kb cDNA insert contained 394 nucleotides of the coding region a long 3' untranslated region and two more poly(A) addition signals (3041 and 3059 nucleotides). An amino acid microsequence surrounding the autophosphorylation site of pure rat ovarian R-II51 agreed with the amino acid sequence deduced from the nucleotide sequence of the cDNA. Northern blot analyses demonstrated two major mRNA species (1.8 and 3.2 kb in size) in hormone-primed rat ovaries which were approximately 10- and 50-fold greater than the R-II mRNA content in rat brain and rat heart, respectively. Southern blot analysis of rat liver DNA indicated that a single gene codes for R-II51 mRNA. Structural differences among rat ovarian R-II51, rat heart R-II54, and the known amino acid sequences of bovine R-II and R-I subunits also indicate that the rat ovarian R-II51 subunit is the product of a distinct gene.     

The complete cDNA sequence of bovine coagulation factor V.

Lack of availability of a primary structure for bovine factor V has hindered detailed analysis of a vast majority of structure-function correlations on this molecule. To determine the primary structure of bovine factor V, we used liver mRNA as a template for the synthesis of three cDNA libraries. The sequences of seven overlapping cDNA clones infer two bovine factor V variants. Variant 1 results in a 6910-basepair (bp) cDNA including 103 bp of 5'-untranslated sequence, 6633 bp of coding sequence and 171 bp of 3'-untranslated sequence with a putative polyadenylation site. Variant 2 differs only in the size of the coding sequence (6618 bp). The open reading frame translates to factor V consisting of 2211 (or 2206) amino acids including a 28-amino acid signal peptide. Comparison of the amino acid sequences with human factor Va reveals 84% identity for the heavy and 86% for the light chains. In contrast, the B domain (connecting region) exhibits only 59% identity relative to the human molecule. The bovine B domain contains two repeats of a 14-amino acid structure that is contained only once in the human sequence. Bovine factor V lacks one of the nine amino acid repeats and one of the 17 amino acid repeats present in the human B domain. Factor V has little homology to the factor VIII molecule in the B domain. The 17-amino acid repeat missing in bovine factor V allows identification of an 18-amino acid sequence that is homologous to the B domain of human factor VIII. These 18 amino acids may either constitute the unique vestige of a divergent evolution between the B domains of factors V and VIII or reveal the convergent evolution toward a critical epitope involved in the activation of both procofactors.     

Molecular cloning of a multifunctional chicken protein acting as the prolyl 4-hydroxylase beta-subunit, protein disulphide-isomerase and a cellular thyroid-hormone-binding protein. Comparison of cDNA-deduced amino acid sequences with those in other species.

Several recent studies indicate that a single polypeptide may act as the beta-subunit of prolyl 4-hydroxylase, the enzyme protein disulphide-isomerase and a cellular thyroid-hormone-binding protein. We report here the isolation and characterization of cDNA clones encoding this multifunctional protein in the chicken. All the coding sequences were determined on the basis of nucleotide sequencing of five cDNA clones and amino acid sequencing of the N-terminal end of the chicken beta-subunit. The processed polypeptide contains 493 amino acid residues, the size of the respective mRNA being about 2.7 kb. The chicken beta-subunit cDNA sequences were 78% homologous to the previously reported human beta-subunit cDNA sequences at the nucleotide level and 85% homologous at the amino acid level. The homology of the chicken beta-subunit sequences to those reported for bovine thyroid-hormone-binding protein and rat protein disulphide-isomerase was also 85% at the amino acid level. Primary-structure comparisons between the four species indicated that the two proposed active sites of protein disulphide-isomerase, the two Trp-Cys-Gly-His-Cys-Lys sequences, are located within highly conserved regions, which are also homologous to the active sites of a number of thioredoxins. The middle of the polypeptide has an additional conserved region 100 amino acid residues in length in which the degree of homology between the four species is 94% at the amino acid level. This long conserved region may also be important for some of the multiple functions of the protein. The four extreme C-terminal amino acids of the polypeptide in all four species are Lys-Asp-Glu-Leu, a sequence that has been suggested to function as a signal for the retention of a protein in the endoplasmic reticulum.     

Cloning and expression of the bovine 11beta-hydroxysteroid dehydrogenase type-2

The bovine 11β-hydroxysteroid dehydrogenase type 2 enzyme (11β-HSD-2) cDNA was cloned from three overlapping PCR fragments using primers based on the human and ovine 11β-HSD-2 cDNA sequences. Both cDNA ends were obtained by a modified RACE (Rapid Amplification of cDNA Ends) method. The bovine 11β-HSD-2 cDNA is 1878 bp long, excluding the poly(A) tail. It consists of a 5′-untranslated region of 133 bp, an open reading frame of 1215 bp and a 3′-untranslated region of 530 bp. Bovine 11β-HSD-2 cDNA is highly homologous to that of the sheep (92%) and less related to the human (67%), rabbit (65%), rat (52%) and mouse (45%) cDNA. The predicted bovine 11β-HSD-2 protein contains 404 amino acid residues with a calculated mol wt of 43,985. It is homologous to the sheep (98%) and human (88%) protein, and less related to that of the rabbit (76%), rat (80%) and mouse (77%). The cloned 11β-HSD-2 cDNA was transfected into CHOP cells and the enzymatic characteristics determined. The enzyme functions primarily as an oxidase, uses NAD⁺ and is more active with corticosterone as a substrate than with cortisol or dexamethasone. It is expressed in high concentrations in kidney, adrenal and colon, and in small concentrations in liver, heart and lung. In conclusion, the 11β-HSD-2 enzyme of cattle is very similar to that of other species in its structure and enzymatic characteristics.     

cDNA cloning and sequencing, gene expression, and immunolocalization of thrombomodulin in the Sprague-Dawley rat.

Thrombomodulin (TM), in addition to its significance in the protein C anticoagulant pathway and cardiovascular diseases, has recently been shown to play important roles in normal embryonic development, several inflammatory conditions, as well as in tumor biology and in the pathogenesis of chronic radiation toxicity. We cloned and sequenced the cDNA encoding the complete TM protein from the Sprague-Dawley rat. The cDNA sequence consisted of a 78-bp 5' non-coding region and a 1731-bp open reading frame encoding 577 amino acids. Comparison of the deduced amino acid sequences showed Sprague-Dawley rat TM to be 87% homologous with mouse and 70.3% with human TM. In addition to the previously described highly conserved region in the lectin-like domain, another region was found which possessed significant homology among the species and may be involved in regulating cell surface expression of TM. Primers and fluorogenic probe for 5' exonuclease-based real time RT-PCR detection (TaqMan PCR) were constructed based on the cDNA sequence information and used to determine steady-state TM mRNA levels in lung, intestine, kidney, brain, and liver. The highest TM mRNA levels were found in lung and the lowest in liver. Immunohistochemistry confirmed that TM was mainly localized on the endothelium of blood vessels and lymphatics. The alveolar capillaries of lung showed the strongest immunoreactivity, whereas the endothelium of hepatic sinusoids and cerebral cortex were virtually negative.     

Human liver fatty acid binding protein cDNA and amino acid sequence. Functional and evolutionary implications

Human liver fatty acid binding protein (L-FABP) cDNA clones were identified in a liver cDNA library. The two longest clones were completely sequenced. The nucleotide sequence predicts a protein of 127 amino acid residues. Identity of the clones was confirmed by limited amino acid sequence analysis of purified human L-FABP peptides and Edman degradation of radiolabeled in vitro translated FABP. Statistical analysis of the amino acid and mRNA sequences of human L-FABP, rat L-FABP, rat intestinal (I-) FABP, and mouse 422 protein indicates that the human and rat L-FABPs are highly homologous and that L-FABP and I-FABP diverged a long time ago (approximately 650-690 million years ago), although they are more closely related to each other than either of them is to 422 protein. Secondary structure predictions from the primary sequence of human and rat L-FABP reveal a region (residues 12-30) that might be the putative fatty acid binding domain of the two L-FABPs. Knowledge of the primary amino acid sequence of L-FABP and possible functional domains will be pivotal in further defining and understanding the mechanism of ligand binding and transfer by this protein.     

Analysis of the human, bovine and rat 33-kDa proteins and cDNA in retina and pineal gland

A monoclonal antibody (mAb) was produced against a bovine retinal 33-kDa protein. Several clones of 33-kDa protein were isolated from each library of cDNA from human, bovine and rat retinas and rat pineal gland by mAb screening and by hybridization with cDNA probes. Each of the four cDNA sequences was determined and amino acid (aa) sequences were deduced from the nucleotide sequences. The latter were nearly identical in rat retina and rat pineal gland (99.6%) and were similar in human, bovine and rat retina (more than 87%). Each of these cDNAs had one long ORF and encoded 245 or 246 aa. The deduced aa sequences in rat retina and rat pineal gland were virtually identical and the sequences in human, bovine and rat retina were highly homologous (more than 88%). The predicted Mr for each of these proteins was 28,246 in the human, 28,176 in bovine, 28,143 in rat retina, and 28,129 in rat pineal gland. Each of the sequences has a putative site for phosphorylation by A kinase; we have confirmed that the putative site is Ser73. These results show that the 33-kDa proteins in the retina and pineal gland have the same sequences and the same phosphorylation site and suggest that the functional role of this protein is the same in the retina and pineal gland.     

白毛黑眼兔眼部虹膜Trp1、Trp2基因克隆与分析

目的 克隆日本大耳白兔白毛黑眼系(白毛黑眼兔)眼部虹膜Trp1、Trp2基因,获取其完整的外显子序列.进一步推测这两个基因编码的蛋白,并分析其特征.方法 从白毛黑眼兔的黑色虹膜组织中提取RNA,并反转录成cDNA.利用来自小鼠、大鼠和人的同源引物,扩增获得白毛黑眼兔Trp1、Trp2基因外显子片段.然后对已知片段进行3' RACE和5'RACE,从而获得白毛黑眼兔Trp1、Trp2基因外显子完整序列.利用相关软件对获得序列进行翻译和分析.结果 首次获得了白毛黑眼兔Trp1、Trp2基因外显子全序列.该实验兔Trp1基因的编码序列全长1604个碱基,其核苷酸序列与人的相似度为87.9％,与小鼠的相似度为82.7％.TRP1成熟蛋白包含513氨基酸,氨基酸序列与人的相似度为89.8％,与小鼠的相似度为86.6％.该实验兔Trp2基因序列全长1554个碱基,其核苷酸序列与人的相似度为83.2％,与小鼠的相似度为81.9％.TRP2成熟蛋白包含494个氨基酸,其序列与人的一致度为84.2％,与小鼠的一致度为84.4％.结论 本研究获得的TRP1、TRP2的序列与已知的家兔酪氨酸相关蛋白家族成员TYR的序列进行比对,结果显示这三种蛋白之间有较高的相似度,并且TRP1和TRP2蛋白序列表现出了酪氨酸酶家族结构上的保守性和特有的结构特征.     

Isolation and sequence analysis of cDNA for rat carboxypeptidase E [EC 3.4.17.10], a neuropeptide processing enzyme

Carboxypeptidase E (CPE) is the carboxypeptidase B-like enzyme associated with the biosynthesis of numerous peptide hormones and neurotransmitters. This enzyme has been previously purified to homogeneity from bovine tissues, and cDNA clones (non-full length) isolated from a bovine pituitary cDNA library. In the present study, cDNA encoding full-length rat CPE has been isolated and sequenced. Both the nucleotide and amino acid sequences of rat CPE show substantial homology with the bovine sequences. The bovine and rat nucleotide sequences are homologous within the entire coding region, as well as within several portions of the 3'-untranslated region. The predicted amino acid sequence of rat CPE is greater than 90% homologous with the bovine enzyme. Northern blot analyses indicate a single species of CPE mRNA approximately 2100 nucleotides in length to be present in many neural and endocrine tissues. High levels of CPE mRNA are present in rat hypothalamus, hippocampus, midbrain, striatum, and cerebral cortex; and moderate levels are present in the brain stem, cerebellum, heart, adrenal, and eye. Low levels are detected in testis and duodenum, but not in liver or thymus. This tissue-specific expression of CPE mRNA is consistent with the proposed role for this enzyme in the production of numerous peptide hormones and neurotransmitters.     

Rat pineal S-antigen: sequence analysis reveals presence of alpha-transducin homologous sequence

S-antigen (S-Ag) is a soluble, highly antigenic protein, the administration of which induces autoimmune uveitis. This protein is found in the retina and pineal. Retinal S-Ag from three species has been sequenced. In this study rat pineal S-Ag was sequenced. Clones were isolated from a rat pineal lambda gt11 cDNA library by probing with a 300 bp fragment of mouse retinal S-Ag cDNA containing the 5'-coding region. The largest clone isolated (RPS-118; 1364 bp) contained the entire coding sequence. Comparison of the rat pineal and mouse retinal S-Ag nucleotide sequences indicated a high homology (95%). The deduced amino acid sequence was found to contain 403 residues (congruent to 44 992 Da). Comparison of the rat pineal and mouse retinal S-Ag amino acid sequences also revealed high homology (97%). The similarity of both the nucleotide and amino acid sequences of rat pineal and mouse retinal S-Ag indicates that expression of the S-Ag gene in both tissues is similar. Further analysis of the rat pineal S-Ag sequence indicated that it contained essentially the same major uveitopathogenic region of S-Ag present in bovine retina; minor uveitopathogenic sites were somewhat different. As is true of retinal S-Ag, rat pineal S-Ag contains the same consensus phosphoryl-binding site present in many GTP/GDP-binding proteins and a homologous sequence found in the C-terminus of alpha-transducin. These sequences may play a role in the action of pineal S-Ag in transmembrane signal transduction.     

Molecular cloning of DNA complementary to bovine growth hormone mRNA

We have cloned DNA complementary to mRNA coding for bovine growth hormone (bGH). Double-stranded DNA complementary to bovine pituitary mRNA was inserted into the Pst I site of plasmid pBR322 by the dC x dG tailing technique and amplified in E. coli x 1776. A recombinant plasmid containing bGH cDNA ws identified by hybridization to cloned rat growth hormone cDNA. It contains the entire coding and 3'-untranslated regions and 31 bases in the 5'-untranslated region. Nucleotide sequence analysis determined the sequence of the 26-amino acid signal peptide and confirmed the published amino acid sequence of the secreted hormone at all but 2 residues. Codon usage is nonrandom, with 81.7% of the codons ending in G or C. The nucleotide sequence of bGH mRNA is 83.9% homologous with rat GH mRNA and 76.5% homologous with human GH mRNA, while the respective amino acid sequence homologies are 83.5% and 66.8%.