Characterization of estrogen receptor beta2 and expression of the estrogen receptor subtypes alpha, beta1, and beta2 in the protandrous black porgy (Acanthopagrus schlegeli) during the sex change process

Estrogens play an important role in many physiological processes in both female and male vertebrates, mediated by specific nuclear receptor, estrogen receptors (ERs). We have isolated a third ER (ERbeta2), which was found to contain 2004 nucleotides including an open reading frame that encodes 667 amino acids. We have also cloned ERalpha and ERbeta1 from the published information (GenBank accession nos. AY074780 and AY074779) and investigated the expression pattern of these ER subtypes in the gonads during gonad sex change of black porgy by quantitative polymerase chain reaction. Maturity stages can be divided into five stages during the sex change process from immature male to female (immature male, mature male, male of mostly testis, male of mostly ovary and mature female). The expression of ERalpha mRNA was highest in the ovary of mature female, followed by the testis of mature male and testicular portion of mostly testis. ERbeta1 expression was higher in the mature testis and ovary than in the gonads of other maturity stages. In contrast to that, ERbeta2 was highest in the ovary of mature female, and significantly lower levels of ERbeta2 expression were observed in the gonads of the other maturity stages. The present study describes the molecular characterization of ERbeta2, and documents the expression changes of three ER subtypes during sex change process of the protandrous black porgy.     

Cloning, expression and chromosomal localization of a human testis 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase gene

6-Phosphofructo-2-kinase/fructose 2,6-bisphosphatase (PFK-2/FBPase-2) is a bifunctional enzyme responsible for the synthesis and breakdown of Fru-2,6-P2, a key metabolite in the regulation of glycolysis. Several genes encode distinct PFK-2/FBPase-2 isozymes that differ in their tissue distribution and enzyme regulation. In this paper, we present the isolation of a cDNA from a human testis cDNA library that encodes a PFK-2/FBPase-2 isozyme. Sequencing data show an open reading frame of 1407 nucleotides that codifies for a protein of 469 amino acids. This has a calculated molecular weight of 54kDa and 97% similarity with rat testis PFK-2/FBPase-2, with complete conservation of the amino acid residues involved in the catalytic mechanism. Fluorescence in-situ hybridization (FISH) localized testis PFK-2/FBPase-2 gene (PFKFB4) in human chromosome 3 at bands p21-p22. A Northern blot analysis of different rat tissues showed the presence of a 2.4-kb mRNA expressed specifically in testis. In mammalian COS-1 cells, the human testis cDNA drives expression of an isozyme with a molecular weight of 55kDa. This isozyme shows clear PFK-2 activity. Taken together, these results provide evidence for a new PFK-2/FBPase-2 gene coding for a human testis isozyme.     

Molecular cloning and characterization of oppo 1: a haploid germ cell-specific complementary DNA encoding sperm tail protein

We isolated a cDNA clone specifically expressed during spermatogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1085 nucleotides and had an open reading frame of 870 nucleotides encoding a putative protein of 290 amino acid residues. Northern blot analysis revealed a 1.2-kilobase mRNA exclusively expressed in the testis in adult mice; the mRNA was first detected late pachytene stage, and expression increased as the animals matured. The protein encoded by the mRNA had a molecular weight of approximately 33 kDa by Western blot analysis, and was localized to occupy the flagella from the connecting piece through the principal piece. We named this newly isolated gene oppo 1, and we suggest that it plays an important role in sperm tail structure and/or sperm movement.     

Quantitative analysis of estrogen receptor proteins in rat ovary

The mRNAs of estrogen receptor beta (ERbeta), and its splice variant, ERbeta2, are abundant in granulosa cells in the ovary. With the use of antibodies, ERbeta protein has also been shown to be abundantly expressed, but to date no ERbeta2 protein has been demonstrated in the ovary. ERbeta2 has a peptide, 18 amino acids in length, inserted into its ligand-binding domain, resulting in a reported 35-fold reduction in its affinity for estrogen (E2). ERalpha, ERbeta1 and ERbeta2 were quantified by Western blotting and by RT-PCR and their cellular localization in the ovary was examined by immunohistochemistry. In 3- and 5-week-old virgin, pregnant, lactating and post-lactating rats, the level of ERalpha protein ranged between 1.6 and 3.8 fmol/microg total protein. That of ERbeta was 8.8-11.2 and of ERbeta2, in the same samples, 4.1-5.9 fmol/microg total protein. ERbeta2 and ERbeta1 proteins were, therefore, present in approximately equal amounts in the ovary throughout the various reproductive stages. The major ERbeta proteins in rat ovary, detected by their molecular weights on Western blots, were ERbeta1-530 and ERbeta2-548 (530+18 amino acids (aa)). Immunohistochemical staining revealed that ERbeta and ERbeta2 were expressed predominantly in granulosa cells of growing follicles, while ERalpha was found only in theca cells. In some theca cells, both ERalpha, ERbeta2 were expressed. The data suggests that in theca cells, where it is co expressed with ERalpha, ERbeta2 could function as a repressor of ERalpha. However, in granulosa cells where no ERalpha is detectable, and where E2 levels are high, ERbeta2, with its low affinity for E2, could be an important sensor through which E2 exerts regulatory control.     

Cloning and characterization of a cDNA encoding type 1 diacylglycerol acyltransferase from sunflower (Helianthus annuus L.)

A full-length cDNA encoding a putative diacylglycerol acyltransferase (DGAT; EC 2.3.1.20) was obtained from sunflower (Helianthus annuus L.) seeds. The 1524-bp open reading frame of this cDNA, designated as HaDGAT1, encodes a protein of 507 amino acids with a molecular mass of 58.5 kDa showing high homology to DGAT1 enzymes of other plants. The protein characters, such as a predicted structure with a long N-terminal hydrophilic domain followed by 9 transmembrane domains, acyl-CoA-binding signature, diacylglycerol (DAG)-binding and putative endoplasmic reticulum retrieval motifs (ER-DIR), also indicated that HaDGAT belongs to the DGAT1 family. HaDGAT1 is expressed in all plant tissues especially in developing seeds. Expression of recombinant HaDGAT1 in yeast showed an 1.76-fold increase of total fatty acids, especially unsaturated fatty acids such as palmitoleic acid (enhanced by 86.6%) and oleic acid (enhanced by 81.6%).     

Molecular cloning of the common carp (Cyprinus carpio) rhodopsin cDNA

A recombinant phage clone containing a 1584 nucleotides rhodopsin cDNA was screened from a carp retinal cDNA library. The inserted DNA consisting of a single open reading frame of 1062 nucleotides at positions 72 to 1133 encodes a 354 amino acid polypeptide. The deduced amino acid sequence of carp rhodopsin showed 95.7, 85.5 and 74.4% identity with that of goldfish, sand goby and lamprey, respectively. The sites of palmitoylation, glycosylation, disulfide bond formation and Schiff base formation in the putative rhodopsin are all conserved.     

Molecular basis for the subtype discrimination of the estrogen receptor-beta-selective ligand,diarylpropionitrile

Although the two subtypes of the human estrogen receptor (ER), ERalpha and ERbeta, share only 56% amino acid sequence identity in their ligand binding domain (LBD), the residues that surround the ligand are nearly identical; nevertheless, subtype-selective ligands are known. To understand the molecular basis by which diarylpropionitrile (DPN), an ERbeta-selective ligand, is able to discriminate between the two ERs, we examined its activity on ER mutants and chimeric constructs generated by DNA shuffling. The N-terminal region of the ERbeta LBD (through helix 6) appears to be fully responsible for the ERbeta selectivity of DPN. In fact, a single ERalpha point mutation (L384M) was largely sufficient to switch the DPN response of this ER to that of the ERbeta type, but residues in helix 3 are also important in achieving the full ERbeta selectivity of DPN. Using molecular modeling, we found an energetically favorable fit for the S-DPN enantiomer in ERbeta, in which the proximal phenol mimics the A ring of estradiol, and the nitrile engages in stabilizing interactions with residues in the ligand-binding pocket of ERbeta. Our findings highlight that a limited number of critical interactions of DPN with the ERbeta ligand-binding pocket underlie its ER subtype-selective character.     

Molecular cloning and characterization of phosphatidylcholine transfer protein-like protein gene expressed in murine haploid germ cells

We have isolated a cDNA clone specifically expressed in spermiogenesis from a subtracted cDNA library of mouse testis. The cDNA consisted of 1392 nucleotides and had an open reading frame of 873 nucleotides encoding a protein of 291 amino acid residues. Computer-mediated homology search revealed that the nucleotide sequence was unique but the deduced amino acid sequence had similarity to mouse phosphatidylcholine transfer protein (PCTP). We named this newly isolated gene PCTP-like protein. Northern blot analysis revealed a 1.4-kilobase mRNA expressed in the testis, kidney, liver, and intestine with the highest level in the testis. Messenger RNA expression in the testis was detected first on Day 23 in postnatal development and then increased up to adulthood. The protein, having a molecular weight of approximately 40 000, was encoded by the mRNA and was detected at the tail of the elongated spermatids and sperm by immunohistochemical staining.     

cDNA cloning and expression of Bauhinia purpurea lectin.

Bauhinia purpurea lectin (BPA) was purified from seeds of B. purpurea alba. The purified lectin was digested with an endoproteinase, Asp-N, or trypsin and then the amino acid sequences of the resultant fragments were analyzed. Furthermore, a cDNA library for BPA was constructed using RNA isolated from germinated Bauhinia purpurea seeds. By gene cloning, the nucleotide sequence of BPA cDNA and its deduced amino acid sequence were analyzed. The cloned BPA cDNA comprised 1,152 nucleotides and the open reading frame of the cDNA encodes a polypeptide of 290 amino acids including a signal peptide composed of 28 amino acids. BPA expressed in Escherichia coli showed a relative molecular mass of 29 kDa on sodium dodecyl sulfate-polyacrylamide gel. On comparison of its sequence with those of other leguminous seed lectins, BPA showed high homology to the others.     

Isolation and characterization of a cDNA clone for a novel serine-rich neutrophil protein.

A cDNA expression library from pig blood neutrophils was immunoscreened with a rabbit antiserum raised against a 32 kDa neutrophil membrane phosphoprotein. Previous work indicated this protein as a component of the superoxide-forming NADPH oxidase enzyme complex (1,2). Only one cDNA clone (B+) was highly positive. The B+ clone contained a 1109 bp insert, with an open reading frame encoding for 284 amino acids. The deduced B+ amino acid sequence contained a 72 amino acid domain with proline and glutamine repeats and two domains extremely enriched with serine residues. The isolated cDNA hybridizes with a 3.1 kb mRNA expressed in pig and human leukocytes.     

绵羊CAST基因2型和4型转录本的克隆及特性分析

钙蛋白酶抑制蛋白(Calpastatin, CAST)是一种内源性的需要Ca²⁺激活的钙蛋白酶抑制剂, 在肌肉组织的蛋白质降解过程中起重要的调节作用。文章利用牛^CAST基因的mRNA序列, 通过逆转录RT-PCR首次克隆获得绵羊CAST基因2型转录本和4型转录本的部分cDNA序列, 并对序列进行生物信息学分析。CAST基因2型转录本的扩增片段为4 385 bp, 完整的开放阅读框为2 361 bp, 编码786个氨基酸; CAST基因4型转录本的扩增片段为1 467 bp, 完整的开放阅读框为1 317 bp, 编码438个氨基酸。CASTⅡ型蛋白序列存在4个保守结构域, CASTⅣ型蛋白序列存在3个保守结构域; 两者的二级结构均以螺旋为主, 富含疏水区域, 其氨基酸序列存在多个磷酸化位点以及蛋白激酶C(Protein kinase C, PKC)的磷酸化位点。通过RT-PCR分析CAST基因2型转录本和4型转录本的组织表达谱, 结果表明CAST基因2型转录本在所检测的10个组织中均表达, CAST基因4型转录本仅在睾丸组织中表达。     

Cloning and identification of a novel cDNA coding thioredoxin-related transmembrane protein 2

Thioredoxin plays an important role in various cellular processes through redox regulation. Here we report the molecular cloning and characterization of one member of the thioredoxin superfamily, designated as TMX2. The TMX2 cDNA consists of 1644 nucleotides and contains an open reading frame encoding a protein of 372 amino acids with a predicted molecular mass of 42.5 kDa and an isoelectric point of 8.94 . The TMX2 protein may possess an N-terminal signal peptide, a potential transmembrane domain, an Myb DNA-binding domain repeat signature, a thioredoxin consensus pattern, an endoplasmic reticulum (ER) membrane retention signal (KKXX-like motif), and a dileucine motif in the tail. Northern blot analysis shows it is widely expressed in human tissues.     

Molecular cloning of estrogen receptor alpha of the Nile crocodile

Estrogens are essential for normal reproductive activity in female and male vertebrates. In female reptiles, they are essential for ovarian differentiation during a critical developmental stage. To understand the molecular mechanisms of estrogen action in the Nile crocodile (Crocodylus niloticus), we have isolated cDNA encoding the estrogen receptor alpha (ERalpha) from the ovary. Degenerate PCR primers specific to ER were designed and used to amplify Nile crocodile cDNA from the ovary. The full-length Nile crocodile ERalpha cDNA was obtained using 5' and 3' rapid amplification cDNA ends (RACE). The deduced amino acid sequence of the Nile crocodile ERalpha showed high identity to the American alligator ERalpha (98%), caiman ER (98%), lizard ER (82%) and chicken ERalpha (92%), although phylogenetic analysis suggested profound differences in the rate of sequence evolution for vertebrate ER sequences. Expression of ERalpha was observed in the ovary and testis of juvenile Nile crocodiles. These data provide a novel tool allowing future studies examining the regulation and ontogenic expression of ERalpha in crocodiles and expands our knowledge of estrogen receptor evolution.     

The complete nuclear estrogen receptor family in the rainbow trout: discovery of the novel ERalpha2 and both ERbeta isoforms

Estrogen hormones interact with cellular ERs to exert their biological effects in vertebrate animals. Similar to other animals, fishes have two distinct ER subtypes, ERalpha (NR3A1) and ERbeta (NR3A2). The ERbeta subtype is found as two different isoforms in several fish species because of a gene duplication event. Although predicted, two different isoforms of ERalpha have not been demonstrated in any fish species. In the rainbow trout (Oncorhynchus mykiss), the only ER described is an isoform of the ERalpha subtype (i.e. ERalpha1, NR3A1a). The purpose of this study was to determine whether the gene for the other ERalpha isoform, ERalpha2 (i.e., NR3A1b), exists in the rainbow trout. A RT-PCR and cloning strategy, followed by screening a rainbow trout BAC library yielded a unique DNA sequence coding for 558 amino acids. The deduced amino acid sequence had a 75.4% overall similarity to ERalpha1. Both the rainbow trout ERbeta subtypes, ERbeta1 [NR3A2a] and ERbeta2, [NR3A2b] which were previously unknown in this species, were also sequenced as part of this study, and the amino acid sequences were found to be very different from the ERalphas (approximately 40% similarity). ERbeta1 and ERbeta2 had 594 and 604 amino acids, respectively, and had 57.6% sequence similarity when compared to one another. This information provides what we expect to be the first complete nuclear ER gene family in a fish. A comprehensive phylogenetic analysis with all other known fish ER gene sequences was undertaken to understand the evolution of fish ERs. The results show a single ERalpha subtype clade, with the closest relative to rainbow trout ERalpha2 being rainbow trout ERalpha1, suggesting a recent, unique duplication event to create these two isoforms. For the ERbeta subtype there are two distinct subclades, one represented by the ERbeta1 isoform and the other by the ERbeta2 isoform. The rainbow trout ERbeta1 and ERbeta2 are not closely associated with each other, but instead fall into their respective ERbeta subclades with other known fish species. Real-time RT-PCR was used to measure the mRNA levels of all four ER isoforms (ERalpha1, ERalpha2, ERbeta1, and ERbeta2) in stomach, spleen, heart, brain, pituitary, muscle, anterior kidney, posterior kidney, liver, gill, testis and ovary samples from rainbow trout. The mRNAs for each of the four ERs were detected in every tissue examined. The liver tended to have the highest ER mRNA levels along with the testes, while the lowest levels were generally found in the stomach or heart. The nuclear ERs have a significant and ubiquitous distribution in the rainbow trout providing the potential for complex interactions that involve the functioning of many organ systems.     

Cloning and expression of antiviral/ribosome-inactivating protein from <Emphasis Type="Italic">Bougainvillea</Emphasis> x<Emphasis Type="Italic">buttiana</Emphasis>

A full-length cDNA encoding ribosome-inactivating/antiviral protein (RIP/AVP)from the leaves of Bougainvillea x buttiana was isolated.The cDNA consisted of 1364 nucleotides with an open reading frame (ORF)of 960 nucleotides encoding a 35.49 kDa protein of 319 amino acids.The deduced amino acid sequence has a putative active domain conserved in RIPs/AVPs and shows a varying phylogenetic relationship to the RIPs from other plant species.The deduced protein has been designated BBAP1 (Bougainvillea x buttiana antiviral protein1).The ORF was cloned into an expression vector and expressed in E.coli as a fusion protein of approximately 78 kDa.The cleaved and purified recombinant BBAP1 exhibited ribosome-inhibiting rRNA N-glycosidase activity,and imparted a high level of resistance against the tobacco mosaic virus (TMV).