Murine transforming growth factor-beta 2 cDNA sequence and expression in adult tissues and embryos

Murine transforming growth factor-beta 2 (TGF-beta 2) cDNAs were isolated from cDNA libraries derived from a differentiated murine embryonic carcinoma cell line, PCC3. The composite cDNA sequence is 4267 nucleotides long, including a 1217 nucleotides 5'-untranslated sequence, and encodes a murine TGF-beta 2 precursor of 414 amino acids with 96% identity to its human counterpart. Several consensus polyadenylation sequences are present in the 1807 nucleotides 3'-untranslated sequence. Five TGF-beta 2 mRNA species are observed in the developing mouse fetus and they show different patterns of expression during development. TGF-beta 2 mRNA expression was also examined in adult mouse tissues, in which four of the five RNA species were observed. TGF-beta 2 mRNAs were present in all adult mouse tissues examined, except liver, and was most abundant in placenta, the male submaxillary gland and lung. The patterns of expression suggest a physiological role for TGF-beta 2 both in embryonic development and in the maintenance of adult tissues.     

Molecular cloning and characterization of a ras p21-like GTP-binding protein (24KG) from rat liver.

We have isolated cDNA clones from a rat liver cDNA library that encode a ras p21-like small GTP-binding protein (24KG) which was purified from the microsomes-Golgi complex fraction of the rat liver. The cloning was accomplished using polymerase chain reaction amplified with a set of oligonucleotide primers which were designed from the partial amino acid sequences for 24KG. The cDNA contained an open reading frame encoding a 216 amino acid protein with a calculated Mr weight of 24,397. This Mr weight was similar to that of the purified 24KG estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The sequence analysis of 24KG revealed that a 24KG cDNA is the rat counterpart of a rab11 cDNA cloned from a Madin-Darby canine kidney cell cDNA library. The 1.0-kilobase 24KG mRNA corresponding to the isolated cDNA was also detected in various rat tissues, such as brain, testis, spleen, and heart.     

Amplification and cloning of the Chinese hamster glutamine synthetase gene.

A Chinese hamster ovary cell line, KG1MS which is resistant to 5 mM methionine sulphoximine overproduces glutamine synthetase. Overproduction of this 42 000 mol. wt. polypeptide is not seen in either parental KG1 or revertant KG1MSC4-0 cell lines which are resistant to 3 microM and 8 microM methionine sulphoximine, respectively. Restriction endonuclease analysis of DNA from KG1MS cells produces a pattern of amplified DNA fragments not seen in parental KG1 or revertant KG1MSC4-0 digests. Hybridization of cDNA probes complementary to KG1MS poly(A) mRNA against Southern blots of KG1MS restriction digests identifies a specific subset within these amplified sequences which is not detected by cDNA probes made from parental KG1 poly(A) mRNA. One 8.2-kb BglII fragment of KG1MS DNA identified by cDNA hybridization has been cloned to produce recombinant pGS-1. mRNA hybrid selected by pGS-1 translates to a 42 000 mol. wt. polypeptide which co-migrates in polyacrylamide gels with the over-produced protein in KG1MS cells and purified glutamine synthetase. pGS-1 also hybridizes to several mRNA species abundant in KG1MSC4-M, but not KG1, poly(A) mRNA extracts. The high level of resistance to methionine sulphoximine shown by KG1MS cells is due to amplification of a DNA sequence of at least 50 kb which contains the coding region for the enzyme glutamine synthetase.     

p53 gene expression is modulated by endocrine disrupting chemicals in the hermaphroditic fish, Kryptolebias marmoratus

The full-length of cDNA of tumour suppressor gene p53 from the self-fertilizing fish Kryptolebias marmoratus (Km-p53) was determined using molecular cloning and rapid amplification of cDNA ends (RACE). The Complete cDNA sequences of K. marmoratus (Km-p53) gene was 1.8 kb in length. K. marmoratus p53 amino acid sequence showed a high degree of homology with the sequences from fishes, amphibians, and mammals. Although basal level of expression of Km-p53 mRNA was low, all the studied tissues showed some level of expression. After exposure of K. marmoratus to endocrine disrupting chemicals (EDCs) such as bisphenol A, 4-nonylphenol, and 4-tert-octylphenol, Km-p53 expression was significantly increased within 3 h of exposure in juveniles. However, expression was down-regulated by exposure to most of the EDCs when measured at 96 h in adult fish. In adult fish, suppressive effect of EDCs was more pronounced in liver as compared to other tissues. These findings suggest that Km-p53 gene would be involved in cellular defense mechanism in early stage of exposure to EDCs and long-term exposure may suppress its expression. It may be possible that the suppression of p53 by EDCs may predispose the host to environmental chemical carcinogenesis.     

Molecular and functional analysis of human natural killer cell-associated neural cell adhesion molecule (N-CAM/CD56).

The neural cell adhesion molecule (N-CAM/CD56) is a member of the Ig supergene family that has been shown to mediate homophilic binding. Several isoforms of N-CAM have been identified that are expressed preferentially in different tissues and stages of embryonic development. To examine the primary structure of N-CAM expressed in leukocytes, N-CAM cDNA were generated by polymerase chain reaction from RNA isolated from normal human NK cells and the KG1a hematopoietic leukemia cell line. The sequence of leukocyte-derived N-CAM cDNA was essentially identical with N-CAM cDNA from human neuroblastoma cells that encode the 140-kDa isoform of N-CAM. Inasmuch as N-CAM is preferentially expressed on human NK cells and a subset of T lymphocytes that mediate MHC-unrestricted cell-mediated cytotoxicity, we examined the potential role of N-CAM in cell-mediated cytotoxicity and heterotypic lymphocyte-tumor cell adhesion. N-CAM loss mutants were established from the human N-CAM+ KG1a leukemia cell line, and N-CAM cDNA was transfected into a human colon carcinoma cell line and murine L cells. Using this panel of mutants and transfectants, it was determined that expression of N-CAM on these target cells does not affect susceptibility to resting or IL-2-activated NK cell-mediated cytotoxicity. Moreover, expression of N-CAM in these transfectants failed to induce homotypic or heterotypic cellular adhesion. Collectively, these studies indicate that homophilic N-CAM interactions probably do not mediate a major role in the cytolytic interaction between NK cells and N-CAM+ tumor cell targets.     

Expression of an early, nonstructural antigen of herpes simplex virus in cell transformed in vitro by herpes simplex virus.

Hyperimmune rabbit antiserum to an early, nonstructural herpes simplex virus type 2 (HSV-2)-induced polypeptide (VP143) reacted in immunofluorescence tests with a variety of cell lines transformed by HSV-2. Cytoplasmic fluorescence was observed in 10 to 50% of HSV-2-transformed cells, whereas no fluorescence was observed in cells transformed by other oncogenic DNA viruses or by a chemical carcinogen. VP143-specific reactivity could be absorbed from anti-VP143 serum with HSV-2-transformed cells but not with cells transformed by other agents. When HSV-2-transformed cells were synchronized in mitosis and examined at various times postmitosis for VP143-specific fluorescence, the expression of VP143 was shown to be cell cycle dependent.     

Striking similarity of murine nectin-1alpha to human nectin-1alpha (HveC) in sequence and activity as a glycoprotein D receptor for alphaherpesvirus entry

A cDNA encoding the murine homolog of human nectin-1alpha (also known as poliovirus receptor-related protein 1 [Prr1] and herpesvirus entry protein C [HveC]) was isolated. The protein encoded by this cDNA proved to be 95% identical in sequence to the human protein and to have similar herpesvirus entry activity. Upon expression of the murine cDNA in hamster cells resistant to alphaherpesvirus entry, the cells became susceptible to the entry of herpes simplex virus types 1 and 2 (HSV-1 and -2), pseudorabies virus, and bovine herpesvirus 1. HSV envelope glycoprotein D (gD), a viral ligand for human nectin-1alpha, is also a ligand for the murine homolog based on evidence that (i) a soluble hybrid protein composed in part of the murine nectin-1 ectodomain bound specifically to purified soluble forms of HSV-1 and HSV-2 gD as demonstrated by enzyme-linked immunosorbent assay, (ii) a soluble hybrid of HSV-1 gD bound to hamster cells expressing murine nectin-1alpha but not to control cells, and (iii) cells expressing both murine nectin-1alpha and one of the alphaherpesvirus gDs were resistant to entry of HSV-1, indicative of interference with entry resulting from interactions of cell-associated gD with the entry receptor. Northern blot analysis revealed that nectin-1 is expressed in most of the mouse tissues examined and at high levels in the brain, skin, and kidneys. Immunocytochemical localization demonstrated the presence of nectin-1 in epithelial cells of the mouse vagina and also in neuronal cells of the central nervous system, suggesting an expression pattern relevant to both infection at a portal of entry and spread of infection to the brain.     

Molecular Cloning, Mapping, and Expression Analysis of the EIF4A2 Gene in Pig

A full-length cDNA clone encoding the eukaryotic translation initiation factor 4A, isoform 2 (EIF4A2), was cloned from the fetal skeletal cDNA library from the pig (Sus scrofa). EIF4A2 is a highly conserved gene for one of the protein-synthesis initiation factors involved in the binding of mRNA to the ribosome. Based on this cDNA sequence, the deduced protein of 407 amino acids contains the characteristic motifs shared by the DEAD-box supergene family. The genomic nucleotide sequence of this gene was determined and a single nucleotide polymorphism located in the 5′ untranslated region was genotyped. The porcine EIF4A2 was expressed in all tissues examined but in variable amounts. The EIF4A2 expression level in muscle was upregulated through embryonic and neonatal development until adult, suggesting that porcine EIF4A2 was possibly involved in translation regulation of other muscle-related genes in muscle formation and development. In addition, we mapped porcine EIF4A2 to q4.1 of SSC13, in agreement with comparative mapping data.     

Ermap, a gene coding for a novel erythroid specific adhesion/receptor membrane protein

Ermap (erythroid membrane-associated protein), a gene coding for a novel transmembrane protein produced exclusively in erythroid cells, is described. It is mapped to murine Chromosome 4, 57 cM distal to the centromere. The initial cDNA clone was isolated from a day 9 murine embryonic erythroid cell cDNA library. The predicted peptide sequence suggests that ERMAP is a transmembrane protein with two extracellular immunoglobulin folds, as well as a highly conserved B30.2 domain and several phosphorylation consensus sequences in the cytoplasmic region. ERMAP shares a high homology throughout the entire peptide with butyrophilin, a glycoprotein essential for milk lipid droplet formation and release. A GFP-ERMAP fusion protein was localized to the plasma membrane and cytoplasmic vesicles in transiently transfected 293T cells. Northern blot analysis and in-situ hybridization demonstrated that Ermap expression was restricted to fetal and adult erythroid tissues. ERMAP is likely a novel adhesion/receptor molecule specific for erythroid cells.     

Identification of development and tissue-specific gene expression in the fathead minnow Pimephales promelas, Rafinesque using computational and DNA microarray methods

The fathead minnow Pimephales promelas serves as a model organism for assessing the effects of environmental contaminants on early life stage growth and development. Yet, the utilization of genomic tools has been hindered by the lack of genome sequence and genomic information known from this model species. Utilizing published cDNA library sequences, the authors used sequence similarity to compare 4105 cDNAs isolated from fathead minnow fry (<14 days old) with over 250 000 adult cDNA sequences derived from whole body and various tissue types. The objectives of the computational subtraction were to (1) assess the extent of sequence similarity between developing and adult cDNA libraries and (2) predict which cDNA clones are expressed only in developing organisms. The results of the computational predictions were assessed through the construction of a development‐specific DNA microarray targeting all 4105 sequences in the fry cDNA library as well as 56 known mRNAs in P. promelas. Gene expression was determined by comparing total RNA isolated from fry with total RNA isolated from adult samples (whole animal, kidney, liver, brain, ovary and testes). The results showed that 1381 of the targeted fry cDNA sequences (34%) displayed expression across all sample comparisons, and of these, only 166 genes were found to harbour fry‐specific expression (i.e. no expression in adult samples). Of note, 69% of the genes computationally predicted to be fry specific were found across all experimental results; yet, only 27% of the computationally predicted fry‐specific sequences were experimentally confirmed to be fry specific. An important result was the identification of many novel mRNA sequences specific to the developing minnow, which lack homology with any other known sequence. In addition, the study results included tissue‐specific expression in adult samples. These results demonstrate the capabilities and limitations of inter‐library sequence comparisons as a predictor of gene activity in non‐sequenced organisms and tissues, as well as DNA microarray gene expression studies in non‐sequenced organisms.     

花椒窄吉丁气味结合蛋白基因AzanOBP3的克隆、原核表达及组织表达谱分析

【目的】本研究克隆花椒窄吉丁Agrilus zanthoxylumi气味结合蛋白(odorant binding protein, OBP)基因AzanOBP3,并对其进行序列和表达分析,旨在更好地了解OBP基因在花椒窄吉丁成虫触角识别气味物质过程中的作用,为农林害虫的绿色防控提供理论依据。【方法】利用RT-PCR扩增花椒窄吉丁气味结合蛋白基因AzanOBP3的cDNA序列,采用生物信息学软件分析其核苷酸和氨基酸序列;构建重组表达载体pET-28a(+)/AzanOBP3,转化到大肠杆菌Escherichia coli BL21(DE3)感受态细胞中进行融合蛋白的IPTG诱导表达,SDS-PAGE及Western blot鉴定重组表达蛋白;基于qPCR技术分析AzanOBP3基因在花椒窄吉丁雌雄成虫不同组织(头、胸、腹、足和翅)中的表达情况。【结果】克隆获得了花椒窄吉丁气味结合蛋白基因AzanOBP3的cDNA全长序列(GenBank登录号:MT318832),预测到其开放阅读框长414 bp,共编码137个氨基酸,等电点为4.79,蛋白分子量为16.038 kD,N末端具有29个氨基...     

Molecular cDNA cloning and analysis of the organization and expression of the IL-1beta gene in the Nile tilapia, Oreochromis niloticus

The full-length cDNA sequence of interleukin-1beta (IL-1beta) from the Nile tilapia, Oreochromis niloticus, was determined by using PCR with primers designed from known fish IL-1beta sequences followed by elongation of the 5' and 3' ends using Rapid Amplification of cDNA Ends (RACE). The cDNA contains a 92-bp 5' untranslated region (UTR), a single open reading frame (ORF) of 732 bp that translates into a 243-amino acid molecule, a 341-bp 3' UTR with four cytokine RNA instability motifs (ATTTA), and a polyadenylation signal (AATAAA) at 15 nucleotides upstream of the poly(A) tail. The organization of the genomic IL-1beta based on the cDNA sequence appeared to be 4 introns and 5 exons. In comparison with known IL-1beta amino acid sequences, including human, catshark, trout, turbot, carp, sea bream, sea bass and goldfish, the amino acid sequence deduced from the cDNA sequence of Nile tilapia showed different levels of identity ranging from 25.32% to 66.80% and homology ranging from 41.88% to 82.19%. Although the entire cDNA sequence of Nile tilapia IL-1beta showed from 49.45% to 67.05% identity to those of other reported IL-1beta cDNAs, each exon also showed different levels of identity to the corresponding exons of other reported IL-1beta cDNAs. The highest nucleotide sequence identity for exon 1 and exons 2-5 of Nile tilapia IL-1beta was found in the corresponding exons of sea bream and sea bass, respectively. After in vitro stimulation with lipopolysaccharide (LPS), we found an increased level of IL-1beta expression in head kidney cells compared to that of unstimulated cells. However, this difference was no longer apparent after 4 h of stimulation, at which time the levels were similar in stimulated and unstimulated cells. Head kidney cells stimulated in vivo by an intraperitoneal injection of LPS showed a peak level of IL-1beta expression after 1 day and a decreased level after 3 days. At 7 days after stimulation, we were hardly able to detect IL-1beta expression.     

The ram: a novel low molecular weight GTP-binding protein cDNA from a rat megakaryocyte library

A novel low Mr GTP-binding protein cDNA was isolated from a rat megakaryocyte cDNA library with a synthetic oligonucleotide probe corresponding to an 8-amino acid sequence specific for c25KG, a GTP-binding protein previously isolated from human platelet cytosol fraction [(1989) J. Biol. Chem. 264, 17000-17005]. The cDNA has an open reading frame encoding a protein of 221 amino acids with a calculated Mr of 25068. The protein is designated as ram (ras-related gene from megakaryocyte) protein (ram p25). The amino acid sequence deduced from the ram cDNA contains the consensus sequences for GTP-binding and GTPase domains. ram p25 shares about 23%, 39% and 80% amino acid homology with the H-ras, smg25A and c25KG proteins, respectively. The 3.5-kb ram mRNA was detected abundantly in spleen cells.     

New epidermal keratin genes from Xenopus laevis: hormonal and regional regulation of their expression during anuran skin metamorphosis

Xenopus larval keratin (XLK) was isolated by gel electrophoresis of proteins of tadpole skin. Screening of an expression cDNA library of tail tissues by specific polyclonal antibodies against XLK produced XLK cDNA (xlk). Its complete nucleotide and predicted amino acid sequences revealed that XLK was a new member of type II keratin. Screening of a cDNA library of adult Xenopus skin using an oligonucleotide probe which had been designed from well-conserved N-terminal amino acid sequences of the rod domain of type I keratin produced two cDNAs, xak-a and xak-b, which were found to be new members of type I keratin gene. Northern blot analysis showed that xlk was expressed exclusively in the larval skin whereas xak-a and xak-b were expressed exclusively in the adult skin. Their expression level was regulated in a region- and metamorphic stage- dependent manner during larval skin development. mRNA in situ hybridization experiments identified the cells that expressed xlk, and xak-a and xak-b as larva- specific epidermal cells (skein cells and basal cells), and adult suprabasal epidermal cells, respectively. These three genes were found to be late responsive to thyroid hormone. Phylogenetic relationships of these keratins with known ones are discussed.     

Herpes simplex virus DNA in transformed cells: sequence complexity in five hamster cell lines and one derived hamster tumor.

Analyses of the hybridization kinetics of labeled herpes simplex virus 2 (HSV-2) DNA with DNA from five hamster cell lines transformed by UV light-irradiated HSV-2 revealed the following. (i) Viral DNA sequences were detected in all five cell lines tested. (ii) None of the cell lines contained the full complement of HSV-2 DNA. (iii) The amount of viral DNA present in the cells varied in different transformed cell lines and ranged from 8 to 32% of the HSV-2 DNA genome in 1 to 3 copies/cell. (iv) Two parallel passages of the same cell line (333-2-29) differed in the amount of viral DNA they contained. We also compared the viral DNA sequences present in (i) one transformed cell line (333-8-9) propagated serially in culture for 80 passages, (ii) a tumor produced by inoculation of a newborn hamster with the 333-8-9 cells, and (iii) a cell line derived from a hamster tumor as above and propagated in culture for 32 passages. The results show that viral DNA present in the hamster tumor and in the cells derived from the tumor had a lower sequence complexity than that present in the original serially passaged 333-8-9 cell line.