Primer protein of bacteriophage M2 exposes the RGD receptor site upon linking the first deoxynucleotide

Summary Using electroporation with the phage PRD1 genome, we set up a high-frequency DNA transfer system for a linear dsDNA molecule with 5-covalently linked terminal proteins. The transfer was saturated when more than 100 ng of PRD1 genome was used. Electroporation efficiency was about four orders of magnitude higher than that obtained with transfection. Removal of the terminal protein abolished plaque formation, which could not be rescued by supplying the terminal protein or phage DNA polymerase or both in trans.     

The Luteinizing Hormone Surge Regulates Circadian Clock Gene Expression in the Chicken Ovary

The molecular circadian clock mechanism is highly conserved between mammalian and avian species. Avian circadian timing is regulated at multiple oscillatory sites, including the retina, pineal, and hypothalamic suprachiasmatic nucleus (SCN). Based on the authors’ previous studies on the rat ovary, it was hypothesized that ovarian clock timing is regulated by the luteinizing hormone (LH) surge. The authors used the chicken as a model to test this hypothesis, because the timing of the endogenous LH surge is accurately predicted from the time of oviposition. Therefore, tissues can be removed before and after the LH surge, allowing one to determine the effect of LH on specific clock genes. The authors first examined the 24-h expression patterns of the avian circadian clock genes of Bmal1, Cry1, and Per2 in primary oscillatory tissues (hypothalamus and pineal) as well as peripheral tissues (liver and ovary). Second, the authors determined changes in clock gene expression after the endogenous LH surge. Clock genes were rhythmically expressed in each tissue, but LH influenced expression of these clock genes only in the ovary. The data suggest that expression of ovarian circadian clock genes may be influenced by the LH surge in vivo and directly by LH in cultured granulosa cells. LH induced rhythmic expression of Per1 and Bmal1 in arrhythmic, cultured granulosa cells. Furthermore, LH altered the phase and amplitude of clock gene rhythms in serum-shocked granulosa cells. Thus, the LH surge may be a mechanistic link for communicating circadian timing information from the central pacemaker to the ovary. (Author correspondence: stischkau@siumed.edu)     

Integrin binding human antibody constant domains--probing the C-terminal structural loops for grafting the RGD motif

Recently, it has been demonstrated that loops of the crystallizable fragment of IgG1 (IgG1-Fc) can be engineered to form antigen-binding sites. In this work C-terminal structural loops in the CH3 domains of homodimeric IgG1-Fc have been functionalized to form integrin-binding sites in order to probe the effect of engineering on structural integrity and thermal stability of IgG1-Fc as well as on binding to the ligands Protein A, CD16 and FcRn, respectively. The peptide sequence GCRGDCL - a disulfide-bridged cyclic heptapeptide that confers binding to human αvβ3 integrin was introduced into AB, CD and/or EF loops and single and double mutants were heterologously expressed in Pichia pastoris. Integrin binding of engineered IgG-Fc was tested using both binding to coated αvβ3 integrin in ELISA or to αvβ3-expressing K562 cells in FACS analysis. Additionally, blocking of αvβ3-mediated cell adhesion to vitronectin was investigated. The data presented in this report demonstrate that bioactive integrin-binding peptide(s) can be grafted on the C-terminal loops of IgG-Fc without impairing binding to effector molecules. Observed differences between the investigated variants in structural stability and integrin binding are discussed with respect to the known structure of IgG-Fc and its structural loops.     

Performance of distance-based DNA barcoding in the molecular identification of Primates

For comparative primatology proper recognition of basal taxa (i.e. species) is indispensable, and in this the choice of a suitable gene with high phylogenetic resolution is crucial. For the goals of species identification in animals, the cytochrome c oxidase subunit 1 (cox1) has been introduced as standard marker. Making use of the difference in intra- and interspecific genetic variation – the DNA barcoding gap – cox1 can be used as a fast and accurate marker for the identification of animal species. For the Order Primates we compare the performance of cox1 (166 sequences; 50 nominal species) in species-identification with that of two other mitochondrial markers, 16S ribosomal RNA (412 sequences, 92 species) and cytochrome b (cob: 547 sequences, 72 species). A wide gap exist between intra- and interspecific divergences for both cox1 and cob genes whereas this gap is less apparent for 16S, indicating that rRNA genes are less suitable for species delimitation in DNA barcoding. For those species where multiple sequences are available there are significant differences in the intraspecific genetic distances between different mitochondrial markers, without, however, showing a consistent pattern. We conclude that cox1 allows accurate differentiation of species and as such DNA barcoding may have an important role to play in comparative primatology.     

Identification of a D-amino acid decapeptide HIV-1 entry inhibitor

Entry of human immunodeficiency virus type 1 (HIV-1) virion into host cells involves three major steps, each being a potential target for the development of entry inhibitors: gp120 binding to CD4, gp120-CD4 complex interacting with a coreceptor, and gp41 refolding to form a six-helix bundle. Using a D-amino acid decapeptide combinatorial library, we identified peptide dC13 as having potent HIV-1 fusion inhibitory activity, and effectively inhibiting infection by several laboratory-adapted and primary HIV-1 strains. While dC13 did not block binding of gp120 to CD4, nor disrupt the gp41 six-helix bundle formation, it effectively blocked the binding of an anti-CXCR4 monoclonal antibody and chemokine SDF-1alpha to CXCR4-expressing cells. However, because R5-using primary viruses were also neutralized, the antiviral activity of dC13 implies additional mode(s) of action. These results suggest that dC13 is a useful HIV-1 coreceptor antagonist for CXCR4 and, due to its biostability and simplicity, may be of value for developing a new class of HIV-1 entry inhibitors.     

Expression of the SNF1 gene from Candida tropicalis is required for growth on various carbon sources, including glucose

SNF1 of Saccharomyces cerevisiae is an essential gene for the derepression of glucose repression. A homolog of SNF1 (CtSNF1) was isolated from an n-alkane-assimilating diploid yeast, Candida tropicalis. CtSNF1 could complement the snf1 mutant of S. cerevisiae. The previously published method for introducing the exogenous DNA into C. tropicalis was employed to construct SNF1/ snf1 heterozygote and snf1/snf1 homozygote strains. The successfully constructed SNF1/snf1 heterozygote was named KO-1. Disruption of the second CtSNF1 allele was unsuccessful, suggesting that CtSNF1 might be essential for cell viability. Therefore, in order to control the expression of CtSNF1, a strain (named KO-1G) in which the promoter region of CtSNF1 was replaced with the GAL10 promoter of C. tropicalis was constructed, and the growth of strains KO-1 and KO-1G was compared with that of the parental strain. The growth of strain KO-1 on glucose, sucrose, or acetate did not differ from the growth of the parental strain, but strain KO-1 showed a slight growth retardation on n-alkane. The growth of strain KO-1G on galactose was normal, but the cells stopped growing when transferred to glucose-, acetate-, or n-alkane-containing medium. Northern blot analysis against mRNA from the n-alkane-grown KO-1G strain demonstrated a close relationship between the presence of CtSNF1 mRNA and the growth of the cells, indicating that CtSNF1 is essential for cell viability. Moreover, mRNA levels of isocitrate lyase, which is localized in peroxisomes of C. tropicalis, were significantly affected by the level of CtSNF1 mRNA. Received: 3 May 1999 / Accepted: 14 July 1999