Isolation and characterization of an elongation factor-1α gene in Porphyra yezoensis (Rhodophyta)

A gene of Porphyra yezoensis, coding for the translation elongation factor 1 (EF-1), was isolated from a P. yezoensis genomic library. The coding of 1347 nucleotides encodes a polypeptide of 449 amino acids which exhibits sequence similarity as the known EF-1. An intron is located in the 5 untranslated region. Comparison of the deduced amino acid sequence showed higher similarity to the Porphyra purpurea EF-1tef-c (97%) than to the P. purpurea EF-1tef-s (61%). The mRNA was detected both in the leafy gametophyte and filamentous sporophyte by RT-PCR. The nucleotide sequence data reported in this paper will appear in the DDBJ/EMBL/GenBank databases under accession number AB098024.     

A strategy to obtain backbone resonance assignments of deuterated proteins in the presence of incomplete amide 2H/1H back-exchange

Replacement of non-exchangeable protons by deuterons has become a standard tool in structural studies of proteins on the order of 30–40 kDa to overcome problems arising from rapid ¹H and ¹³C transverse relaxation. However, ¹H nuclei are required at exchangeable sites to maintain the benefits of proton detection. Protein expression in D₂O-based media containing deuterated carbon sources yields protein deuterated in all positions. Subsequent D/H-exchange is commonly used to reintroduce protons in labile positions. Since this strategy may fail for large proteins with strongly inhibited exchange we propose to express the protein in fully deuterated algal lysate medium in 100% H₂O. As a side-effect partial C protonation occurs in a residue-type dependent manner. Samples obtained by this protocol are suitable for complementary ¹H^N- and ¹H-based triple resonance experiments allowing complete backbone resonance assignments in cases where back-exchange of amide protons is very slow after expression in D₂O and refolding of chemically denatured protein is not feasible. This approach is explored using a 35-kDa protein as a test case. The degree of C protonation of individual amino acids is determined quantitatively and transverse relaxation properties of ¹H^N and ¹⁵N nuclei of the partially deuterated protein are investigated and compared to the fully protonated and perdeuterated species. Based on the deviations of assigned chemical shifts from random coil values its solution secondary structure can be established.     

Comparative analysis of IFN-gamma B7.1 and antisense TGF-beta gene transfer on the tumorigenicity of a poorly immunogenic metastatic mammary carcinoma

Cancer progression is attributed in part to immune evasion strategies that include lack of co-stimulation, down-regulation of cell surface MHC molecules, and secretion of immunosuppressive factors, such as transforming growth factor-beta (TGF-beta). Gene therapy has been employed to counter these mechanisms of immune evasion by transference of B7.1, IFN-gamma or antisense TGF-beta genes into tumor cells, resulting in cell surface expression of B7.1, upregulation of MHC class I and class II molecules, or elimination of tumor-derived TGF-beta, respectively. Although each of these transgenes has been shown to alter tumorigenicity in murine models, a direct comparison of their efficacy has not been performed. In this study, we have employed a very aggressive, poorly immunogenic and highly metastatic mammary model, 4T1, to compare the efficacy of B7.1, IFN-gamma and antisense TGF-beta gene transfer in stimulating an anti-tumor response. We demonstrate that both IFN-gamma and antisense TGF-beta gene expression significantly reduced the tumorigenicity of these cells compared to mock transduced cells, with IFN-gamma having a greater effect. In contrast, B7.1 gene transfer did not affect the tumorigenicity of 4T1 cells. The anti-tumor response directed against antisense TGF-beta-expressing 4T1 tumors was mediated by CD4+ and CD8+ T cells. However, CD8+ T cells, and not CD4+ T cells, appeared to mediate the anti-tumor response against IFN-gamma-expressing tumors. Treatment of tumor-bearing animals with IFN-gamma or antisense TGF-beta gene-modified tumor cell vaccines reduced the number of clonogenic metastases to the lungs and liver compared to treatment with mock-transduced cells. Finally, in a residual disease model in which the primary tumor was excised and mice were vaccinated with irradiated tumor cells, treatment of mice with vaccinations consisting of 4T1 cells expressing both antisense TGF-beta and IFN-gamma genes was the most effective in prolonging survival.     

Deuterium isotope effects on the central carbon metabolism of Escherichia coli cells grown on a D2O-containing minimal medium

In this paper it is demonstrated that cross-correlated time modulation of isotropic chemical shifts (`conformational exchange') leads to differential relaxation of double- and zero-quantum coherences, respectively. Quantitative information can be obtained from the time dependence of the interconversion between the two two-spin coherences 2I_xS_x and 2I_yS_y, induced by the differential relaxation. The effect is illustrated with an application to ¹³C,¹⁵N-labeled quail CRP2(LIM2), by studying ¹⁵N-¹H^N multiple-quantum relaxation. Significant cross-correlated fluctuations of isotropic chemical shifts were observed for residues which are part of a disordered loop region connecting two -strands in CRP2(LIM2). Differential ¹H^N and ¹⁵N exchange contributions to multiple-quantum relaxation observed at these sites illustrate the complex interplay between hydrogen bonding events and conformational reorientations in proteins.     

Distinct expression patterns for two Xenopus Bar homeobox genes

The BarH1 and BarH2 homeobox genes are coexpressed in cells of the fly retina and in the central and peripheral nervous systems. The fly Bar genes are required for normal development of the eye and external sensory organs. In Xenopus we have identified two distinct vertebrate Bar-related homeobox genes, XBH1 and XBH2. XBH1 is highly related in sequence and expression pattern to a mammalian gene, MBH1, suggesting that they are orthologues. XBH2 has not previously been identified but is clearly related to the Drosophila Bar genes. During early Xenopus embryogenesis XBH1 and XBH2 are expressed in overlapping regions of the central nervous system. XBH1, but not XBH2, is expressed in the developing retina. By comparing the expression of XBH1 with that of hermes, a marker of differentiated retinal ganglion cells, we show that XBH1 is expressed in retinal ganglion cells during the differentiation process, but is down-regulated as cells become terminally differentiated. Received: 12 August 1999 / Accepted: 5 October 1999     

ASW: a gene with conserved avian W-linkage and female specific expression in chick embryonic gonad

Vertebrates exhibit a variety of sex determining mechanisms which fall broadly into two classes: environmental or genetic. In birds and mammals sex is determined by a genetic mechanism. In mammals males are the heterogametic sex (XY) with the Y chromosome acting as a dominant determiner of sex due to the action of the testis-determining factor, SRY. In birds females are the heterogametic sex (ZW); however, it is not known whether the W chromosome carries a dominant ovary-determining gene, or whether Z chromosome dosage determines sex. Using an experimental approach, which assumes only that the sex-determining event in birds is accompanied by sex-specific changes in gene expression, we have identified a novel gene, ASW (Avian Sex-specific W-linked). The putative protein for ASW is related to the HIT (histidine triad) family of proteins. ASW shows female-specific expression in genital ridges and maps to the chicken W chromosome. In addition, we show that, with the exception of ratites, ASW is linked to the W chromosome in each of 17 bird species from nine different families of the class Aves. Received: 18 October 1999 / Accepted: 10 January 2000     

The noncompetitive inhibitor WW781 senses changes in erythrocyte anion exchanger (AE1) transport site conformation and substrate binding

WW781 binds reversibly to red blood cell AE1 and inhibits anion exchange by a two-step mechanism, in which an initial complex (complex 1) is rapidly formed, and then there is a slower equilibration to form a second complex (complex 2) with a lower free energy. According to the ping-pong kinetic model, AE1 can exist in forms with the anion transport site facing either inward or outward, and the transition between these forms is greatly facilitated by binding of a transportable substrate such as Cl(-). Both the rapid initial binding of WW781 and the formation of complex 2 are strongly affected by the conformation of AE1, such that the forms with the transport site facing outward have higher affinity than those with the transport site facing inward. In addition, binding of Cl(-) seems to raise the free energy of complex 2 relative to complex 1, thereby reducing the equilibrium binding affinity, but Cl(-) does not compete directly with WW781. The WW781 binding site, therefore, reveals a part of the AE1 structure that is sensitive to Cl(-) binding and to transport site orientation, in addition to the disulfonic stilbene binding site. The relationship of the inhibitory potency of WW781 under different conditions to the affinities for the different forms of AE1 provides information on the possible asymmetric distributions of unloaded and Cl(-)-loaded transport sites that are consistent with the ping-pong model, and supports the conclusion from flux and nuclear magnetic resonance data that both the unloaded and Cl(-)-loaded sites are very asymmetrically distributed, with far more sites facing the cytoplasm than the outside medium. This asymmetry, together with the ability of WW781 to recruit toward the forms with outward-facing sites, implies that WW781 may be useful for changing the conformation of AE1 in studies of structure-function relationships.     

The genomic organization and evolution of the natural killer immunoglobulin-like receptor (KIR) gene cluster

Natural killer (NK) immunoglobulin-like receptors (KIRs) are a family of polymorphic receptors which interact with specific motifs on HLA class I molecules and modulate NK cytolytic activity. In this study, we analyzed a recently sequenced subgenomic region on chromosome 19q13.4 containing eight members of the KIR receptor repertoire. Six members are clustered within a 100-kb continuous sequence. These genes include a previously unpublished member of the KIR gene family 2DS6, as well as 2DL1, 2DL4, 3DL1, 2DS4, 3DL2, from centromere to telomere. Two additional KIR genes, KIRCI and 2DL3, which may be located centromeric of this cluster were also analyzed. We show that the KIR genes have undergone repeated gene duplications. Diversification between the genes has occurred postduplication primarily as a result of retroelement indels and gene truncation. Using pre- and postduplication Alu sequences identified within these genes as evolutionary molecular clocks, the evolution and duplication of this gene cluster is estimated to have occurred 30–45 million years ago, during primate evolution. A proposed model of the duplication history of the KIR gene family leading to their present organization is presented. Received: 25 November 1999 / Revised: 10 January 2000