Cloning,characterisation and expression of the α-tubulin genes of the leech,Hirudo medicinalis

We have isolated two α-tubulin cDNAs from the leech, Hirudo medicinalis. Both encode putative proteins of 451 amino-acids which differ from each other at only two positions. Southern blotting suggests that there are only two α-tubulin genes in the leech. The genes contain two introns and, because of the extremely high homology of the nucleotide sequence from the second intron to the end of the genes, we have inferred that a gene conversion event about 9.5 million years ago has homogenised the Hirudo α-tubulin sequences. Using in situ hybridisation to tissue sections, we have shown that the two genes are probably expressed in all neurons of the leech ganglia and that their spatial distribution remains unchanged during neuronal regeneration. The deduced amino-acid sequences of the leech α-tubulins show that they have greatest similarity to those from a platyhelminth, echiuran and mollusc with rather less to arthropod α-tubulins. The protein sequences of the leech α-tubulins have been compared with representatives of those from across all phyla to determine if any specific feature labels certain isotypes of tubulin for neuronal expression.     

Cloning, structural analysis, and expression of the pig IgE ε chain

 As a step in the evolutionary studies of immunoglobulin E (IgE) and for the purpose of developing new reagents that will facilitate a more detailed analysis of IgE-mediated inflammatory reactions in a large animal model, we here present the cloning of the ε chain of IgE in the domestic pig (Sus scrufa). A partial cDNA clone for the ε chain of pig IgE was isolated by polymerase chain reaction (PCR) amplification using degenerate primers directed against conserved regions in the second (CH2) and the fourth (CH4) constant domains of IgE. cDNA derived from mRNA isolated from the spleen and lymph nodes of a pig actively sensitized with a protein extract from the nematode Ascaris suum was used as template. Screening of a spleen cDNA library with the partial cDNA clone as probe resulted in isolation of a clone that contained the entire coding region. The nucleotide sequence was determined and was found to conform with the previously identified mammalian ε-chain sequences. The highest degree of similarity was found to sheep IgE. A DNA construct encoding a baculovirus signal sequence, a histidine hexapeptide, and the CH2-CH3-CH4 domains of the pig IgE ε chain was obtained by PCR amplification. The construct was ligated into the baculovirus expression vector pVL1392. Infection of High Five insect cells with recombinant baculovirus resulted in expression and secretion of a soluble 6 × His-CH2-CH3-CH4 protein product. Received: 9 April 1997 / Revised: 28 May 1997     

Lethality of inducible,meristem-localized ectopic β-glucuronidase expression in plants

GUSA fromEscherichia coli, encoded by theuidA gene, has been successfully used as a plant reporter system for more than a decade with no reported deleterious effects. However, when expressed in coordination with a UDP-glucuronosyltransferase isolated from the root cap meristem ofPisum sativum (PsUGT1) at the onset of mitosis, GUSA expression was lethal in pea, alfalfa, andArabidopsis thaliana. These unexpected results indicate that, under some circumstances, using GUSA in plants is incompatible with life and suggest that the cell-specific lethal phenotype might be useful in selecting for genes specifically involved in regulating the G2-M phase of the cell cycle.     

Cloning,expression and antiviral bioactivity of Red-crowned Crane interferon-α

Interferon-α (IFN-α) genes have been cloned from a variety of animals, but information regarding crane IFN-α has not been reported to date. In this study, we cloned a full-length Red-crowned Crane interferon-α (crIFN-α) gene sequence consisting of a 486 bp partial 5′ UTR, 741 bp complete ORF and 559 bp partial 3′ UTR. This gene encodes a protein of 246 amino acids and shares 60 to 80% identity with avian IFN-α and less than 45% identity with mammalian IFN-α. The expression of crIFN-α with an N-terminal His-tag was investigated in Escherichia coli, and the protein was purified on a nickel column. To obtain activated proteins, crIFN-α inclusion bodies were renatured by dialysis. In vitro cytopathic inhibition assays indicated that the recombinant crIFN-α could inhibit the replication of vesicular stomatitis virus in chicken fibroblasts. These antiviral activities were abrogated by rabbit anti-crIFN-α antibodies in vitro. In addition, an immunofluorescence assay indicated that crIFN-α could be expressed in chicken fibroblasts and was primarily located in the cytoplasm. Taken together, our results suggest that the crIFN-α gene may play an important role in inhibiting the replication of viruses.     

Molecular cloning,expression and characterization of Pekin duck interferon-λ

Interferons (IFNs) are the first line of defense against viral infections in vertebrates. Type III interferon (IFN-λ) is recognized for its key role in innate immunity of tissues of epithelial origin. Here we describe the identification of the Pekin duck IFN-λ ortholog (duIFN-λ). The predicted duIFN-λ protein has an amino acid identity of 63%, 38%, 37% and 33% with chicken IFN-λ and human IFN-λ3, IFN-λ2 and IFN-λ1, respectively. The duck genome contains a single IFN-λ gene that is comprised of five exons and four introns. Recombinant duIFN-λ up-regulated OASL and Mx-1 mRNA in primary duck hepatocytes. Our observations suggest evolutionary conservation of genomic organization and structural features implicated in receptor binding and antiviral activity. The identification and expression of duIFN-λ will facilitate further study of the role of type III IFN in antiviral defense and inflammatory responses of the Pekin duck, a non-mammalian vertebrate and pathogen host with relevance for human and animal health.     

Recombinant expression, purification, and characterization of scorpion toxin BmαTX14

Long-chain and cysteine-rich scorpion toxins exhibit various pharmacological profiles for different voltage-gated sodium channel subtypes. However, the exploration of toxin structure-function relationships has progressed slowly due to the difficulty of obtaining synthetic or recombinant peptides. We now report that we have established an effective expression and purification approach for the novel scorpion toxin BmαTX14. BmαTX14 was over-expressed as inclusion bodies in Escherichia coli. The insoluble pellet was successfully transformed into active peptide by using a refolding procedure. One-step purification by reverse-phase HPLC was sufficient to generate chromatographically pure peptide. The yield of recombinant toxin reached 4mg from 1L LB medium. The pharmacological data further showed that BmαTX14 selectively inhibited the fast inactivation of mNa(v)1.4 (EC(50)=82.3±15.7nM) rather than that of rNa(v)1.2 (EC(50)>30μM), which indicates that BmαTX14 is a new α-like toxin. This work enables further structural, functional, and pharmacological studies of BmαTX14 and similar toxins.     

Ectopic expression of β-lactoglobulin transgenes

Genomic constructs comprising the ovine β-lactoglobulin gene are expressed in a position-independent manner in the mammary gland of transgenic mice. In some lines however, constitutive low-level transgene expression was detected in all other tissues. This ectopic expression presumably represents a position-dependent phenomenon since it was observed in only a proportion (40%) of the lines generated. Different lines of BLG transgenic mice displayed similar temporal patterns of ectopic expression. This pattern differed from that of BLG in the mammary gland. These data imply that the DNA elements that direct position-independent expression of β-lactoglobulin transgenes in the mammary gland do not have the ability to insulate them from position effects in other tissues. Furthermore, the relatively high frequency and constitutive nature of ectopic expression suggests that transgene integration may not be totally random.     

Cloning,expression, and antiviral activity of interferon β from the Chinese microbat,Myotis davidii

Bats are natural reservoir hosts for many viruses that produce no clinical symptoms in bats.Therefore, bats may have evolved effective mechanisms to control viral replication. However, little information is available on bat immune responses to viral infection. Type I interferon(IFN) plays a key role in controlling viral infections. In this study, we report the cloning, expression, and biological activity of interferon β(IFNβ) from the Chinese microbat species, Myotis davidii. We demonstrated the upregulation of IFNB and IFN-stimulated genes in a kidney cell line derived from M. davidii after treatment with poly I:C or infection with Sendai virus. Furthermore, the recombinant IFNβ inhibited vesicular stomatitis virus and bat adenovirus replication in cell lines from two bat species, M. davidii and Rhinolophus sinicus. We provide the first in vitro evidence of IFNβ antiviral activity in microbats, which has important implications for virus interactions with these hosts.     

The cloning, expression, purification, characterization and modeled structure of Caulobacter crescentus β-Xylosidase I

The xynB1 gene (CCNA 01040) of Caulobacter crescentus that encodes a bifunctional enzyme containing the conserved β-Xylosidase and α-L: -Arabinofuranosidase (β-Xyl I-α-L: -Ara) domains was amplified by PCR and cloned into the vector pJet1.2Blunt. The xynB1 gene was subcloned into the vector pPROEX-hta that produces a histidine-fused translation product. The overexpression of recombinant β-Xyl I-α-L: -Ara was induced with IPTG in BL21 (DE3) and the resulting intracellular protein was purified with pre-packaged nickel-Sepharose columns. The recombinant β-Xyl I-α-L: -Ara exhibited a specific β-Xylosidase I activity of 1.25?U?mg(-1) to oNPX and a specific α-L: -Arabinofuranosidase activity of 0.47?U?mg(-1) to pNPA. The predominant activity of the recombinant enzyme was its β-Xylosidase I activity, and the enzymatic characterization was focused on it. The β-Xylosidase I activity was high over the pH range 3-10, with maximal activity at pH 6. The enzyme activity was optimal at 45?°C, and a high degree of stability was verified over 240?min at this temperature. Moreover, β-Xylosidase activity was inhibited in the presence of the metals Zn(2+) and Cu(2+), and the enzyme exhibited K(M) and V(Max) values of 2.89?±?0.13?mM and 1.4?±?0.04?μM?min(-1) to oNPX, respectively. The modeled structure of β-xylosidase I showed that its active site is highly conserved compared with other structures of the GH43 family. The increase in the number of contact residues responsible for maintaining the dimeric structure indicates that this dimer is more stable than the tetramer form.     

Cloning of cDNA,expression, and chromosomal location of genes encoding the three types of subunits of the barley tetrameric inhibitor of insect α-amylase

Three cDNA clones from barley developing endosperm, corresponding to proteins BTAI-CMa, BTAI-CMb and BTAI-CMd, which are the three types of subunits of the tetrameric inhibitor of insect -amylases, have been identified and sequenced. The deduced amino acid sequence of BTAI-CMb corresponds to the CM16/CM17 type of subunit in wheat (92/90% identical residues) and has one putative N-glycosylation site (NLT) and a possible kinase-C phosphorylation site (SCR). The BTAI-CMa sequence differs at four amino acid residues from a previously reported one from cv. Bomi and the sequence deduced for BTAI-CMd completes (11 N-terminal residues) and confirms previously available data. The gene for BTAI-CMa (Iat1) is located in the arm of barley chromosome 7H (syn.1), while genes for both BTAI-CMb (Iat2) and BTAI-CMd (Iat3) are in the long arm of chromosome 4H. The three genes are expressed in endosperm and their mRNAs are not detected in the other tissues tested, except Iat1, which seems to be expressed at a low level in coleoptile and roots, where it is switched off by 50 M methyl jasmonate.     

Two β-tubulin genes,TUB1 and TUB8, of Arabidopsis exhibit largely nonoverlapping patterns of expression

Chimeric reporter genes were used to investigate the patterns of expression of two -tubulin genes, TUB1 and TUB8, in Arabidopsis thaliana. The TUB1 chimeric gene was preferentially expressed in epidermal and cortical cells of primary roots, whereas the TUB8 chimeric gene was preferentially expressed in the endodermal and phloem cells of primary roots and in the vascular tissues of leaves, stems, and flowers of transgenic plants.     

Do the constraints of human speciation cause expression of the same set of genes in brain, testis, and placenta?

Evolution appears to be especially rapid during speciation, and the genes involved in speciation should be evident in species such as humans that have recently speciated or are presently in the process of speciation. Haldane's rule is that when one sex is sterile or inviable in interspecific F(1) hybrids, it is usually the heterogametic sex. For mammals, this implicates genes on the X chromosome as those particularly responsible for speciation. A preponderance of sex- and reproduction-related genes on the X chromosome has been shown repeatedly, but also mental retardation genes are more frequent on the X chromosome. We argue that brain, testis, and placenta are those organs most responsible for human speciation. Furthermore, the high degree of complexity of the vertebrate genome demands coordinate evolution of new characters. This coordination is best attained when the same set of genes is redeployed for these new characters in the brain, testis, and placenta.     

Gene expression of thrombomodulin,TNF-α and NF-KB in coronary artery disease patients of Pakistan

Thrombomodulin (THBD) is an endothelial surface glycoprotein receptor, having a pivotal role in maintaining laminar blood flow. It functions to protect endothelial integrity by exhibiting anti-coagulation and anti-inflammatory properties thereby playing a key role in cardiovascular disease (CVD) pathology. Cholesterol lowering drugs have shown to alter the anti-inflammatory effects of cytokines. Understanding the molecular aspects of THBD gene and its relation to inflammatory cytokines is important to identify new prognostic and therapeutic targets for the CVD treatments. The present study was conducted to measure the expression of THBD, TNF-α and NF-kB genes in coronary artery disease patients (CAD) in Pakistani population. Lipid profile and BMI was compared both on fifty CAD patients and fifty healthy individuals. Expression analysis for THBD, TNF-α and NF-kB was carried out using real time PCR. The effect of lipid lowering drugs on cardiometabolic risk variables especially gene expression was analyzed. Our results indicated that the difference in BMI was marginal; however LDL-cholesterol and triglycerides levels in CAD patients were significantly higher than healthy individuals. THBD gene was significantly up-regulated whereas TNF-α and NF-kB were significantly down regulated in CAD individuals. Further exploration revealed that these variations were accounted to the use of statins by the patients. The use of statins by CAD patients up-regulated the mRNA expression of THBD by down-regulation of inflammatory mediators. The enhanced expression of endothelial THBD in response to cholesterol lowering drugs establishes a novel pleiotropic target that can be of clinical significance in thromboembolic and inflammatory disorders.