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PubMed1996

Covalent attachment of Arc repressor subunits by a peptide linker enhances affinity for operator DNA.

By designing a recombinant gene containing tandem copies of the arc coding sequence with intervening DNA encoding the linker sequence GGGSGGGTGGGSGGG, the two subunits of the P22 Are repressor dimer have been covalently linked to form a single-chain protein called Arc-L1-Arc. The 15-residue linker joins the C-terminus of one monomer to the N-terminus of the second, a distance of approximately 45 A in the Arc-operator cocrystal structure. Arc-L1-Arc is expressed at high levels in Escherichia coli, with no evidence of degradation or proteolytic clipping of the linker, and is more active than wild-type Arc in repression assays. The purified Arc-L1-Arc protein has the molecular weight expected for the designed protein and unfolds cooperatively, reversibly, and with no concentration dependence in thermal-denaturation studies. Arc-L1-Arc protects operator DNA in a manner indistinguishable from that of wild-type Arc in DNase I and copper-phenanthroline footprinting studies, but the covalent attachment of the two monomers results in enhanced affinity for operator DNA. Arc-L1-Arc binds operator DNA half-maximally at a concentration of 1.7 pM, compared with the wild-type value of 185 pM, and also binds DNA fragments containing the left or right operator half-sites more tightly than wild type. Because wild-type Arc is monomeric at sub-nanomolar concentrations and must dimerize before binding to the operator, it was anticipated that Arc-L1-Arc would exhibit a lower half-maximal binding concentration. However, even when the change from a monomeric to a dimeric species is taken into account, the affinity of Arc-L1-Arc for operator and half-operator DNA is greater than the wild-type affinity. This tighter binding appears to result from slower dissociation, as Arc-L1-Arc DNA complexes with full or half-site operators dissociate at rates 5-10 times slower than the corresponding Arc--DNA complexes. Hence, the activity of the designed Arc-L1-Arc protein is substantially increased relative to wild-type Arc in a variety of assays.

Amino Acid Sequence↗

PubMed2006

Seabream ghrelin: cDNA cloning, genomic organization and promoter studies.

Recent studies have indicated that ghrelin stimulates growth hormone release from the pituitary via the growth hormone secretagogue receptor (GHSR). We have previously isolated two GHSR subtypes from the pituitary of the black seabream Acanthopagrus schlegeli. In the present study, we have cloned and characterized ghrelin from the same fish species at both the cDNA and gene levels. The full-length seabream ghrelin cDNA, isolated from sea-bream stomach using a novel approach by exploiting a single conserved region in the coding region, was found to encode a prepropeptide of 107 amino acids, with the predicted mature ghrelin peptide consisting of 20 amino acids (GSSFLSPSQKPQNRGKSSRV). Embedded in this full-length cDNA is a putative fish orthologue of the recently reported mammalian obestatin peptide. The ghrelin gene in black seabream, obtained by genomic PCR, was found to encompass four exons and three introns, possessing the same structural organization as in tilapia and goldfish, but different from that in rainbow trout. In addition, a 2230-bp 5'-flanking region of the seabream ghrelin gene was obtained by genome walking. Sequence analysis revealed that, as in the case of the human ghrelin gene, there is neither a GC box nor a CAAT box present in the isolated 5'-flanking region. However, a number of putative transcription factor-binding sites different from the human counterpart were found in the 5'-flanking region of the seabream ghrelin gene, suggesting that different cis- and trans-acting elements are involved in controlling their gene expression. Functional activity of this 5'-flanking region was examined by cloning it into the pGL3-Basic vector upstream of the luciferase reporter gene and transfected into various cell lines. Positive promoter activity could only be recorded in the colon-derived Caco-2 cells, suggesting that the cloned 5'-flanking region represents the functional promoter of the seabream ghrelin gene, which exhibits tissue-specific promoter activity. Using reverse transcriptase PCR analysis, expression of ghrelin was detected only in the seabream stomach, but not in the other tissues examined, including the brain, gill, intestine, kidney, liver and spleen. This stomach-specific expression of ghrelin in seabream is subject to regulation, as administration of growth hormone or ipamorelin to the fish in vivo was demonstrated to enhance its expression. Reminiscent of the homologous upregulation found in the transcriptional control of the seabream GHSR gene, a similar homologous regulatory mechanism might also exist in controlling the expression of seabream ghrelin. The identification of both GHSR and ghrelin from a single fish species would facilitate our subsequent studies on the elucidation of the physiological functions of the ghrelin/GHSR system in teleost. The possible existence of obestatin in teleost opens up new research avenues on the somatotropic axis in fish.

Amino Acid Sequence↗

PubMed1991

Carboxy-terminal truncated rhuIFN-gamma with a substitution of Gln133 or Ser132 to leucine leads to higher biological activity than in the wild type.

The biological function of the 20 C-terminal amino acids of human interferon-gamma (IFN-gamma) was examined by recombinant DNA methodology. Six truncated IFN-gamma analogues were produced by modification of the 3' end of the coding sequence of the cloned gene, insertion into a vector with the trc promoter and expression of the recombinant IFN-gamma analogue genes in Escherichia coli strain JM 105. The IFN-gamma analogue proteins were shortened by 10 (C-10L), 11 (C-11, C-11L), 14 (C-14L), 19 (C-19L) and 20 (C-20) amino acid. Four of these constructs were modified to have a C-terminal leucine. The expression rates of precipitating IFN-gamma variants in E. coli cells (wild type, C-10L, C-11, C-11L) amount to 35-40% of the total protein, in contrast to 14-21% for the mainly soluble ones (C-14L, C-20). The variant C-19L has an exceptional position in its solubility behaviour with a nearly 1:1 distribution between its soluble and insoluble form by an expression rate of 8%. The purification protocol of the insoluble variants contains a denaturing and a renaturation step. The characteristic step for purification soluble IFN-gamma is HPLC cation-exchange chromatography. The antiviral activities of the variants lacking 14 or more amino acids are less than 2% of the wild-type activity. The variants C-10L, C-11 and C-11L show higher biological activities than wild-type IFN-gamma. The most active variant, C-10L, with leucine as the last C-terminal amino acid, has a fourfold higher specific antiviral activity (A549 cells, encephalomyocarditis virus). Removal, but not replacement of the leucine, represented by the variant C-11, reduces the biological activity compared with variant C-10L. The activity of C-11 is, nevertheless, higher than in the wild type. Comparing the secondary structures, as judged by CD analyses, no significant differences for C-10L, C-14L and C-20, compared with wild type, are observed. Also, all molecules, including the wild-type protein, exist as dimers under physiological conditions. There is a correlation between the grade of truncation and the pI values, which range from pI = 10.4 (wild type) to pI = 8.0 (C-20). The variant C-10L demonstrates a higher temperature stability (tm = 55 degrees C) compared with wild type (tm = 53 degrees C). Perhaps this higher stability will result in a longer half-life in vivo.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗