Three hamster species with different scrapie incubation times and neuropathological features encode distinct prion proteins.

Given the critical role of the prion protein (PrP) in the transmission and pathogenesis of experimental scrapie, we investigated the PrP gene and its protein products in three hamster species, Chinese (CHa), Armenian (AHa), and Syrian (SHa), each of which were found to have distinctive scrapie incubation times. Passaging studies demonstrated that the host species, and not the source of scrapie prions, determined the incubation time for each species, and histochemical studies of hamsters with clinical signs of scrapie revealed characteristic patterns of neuropathology. Northern (RNA) analysis showed the size of PrP mRNA from CHa, AHa, and SHa hamsters to be 2.5, 2.4, and 2.1 kilobases, respectively. Immunoblotting demonstrated that the PrP isoforms were of similar size (33 to 35 kilodaltons); however, the monoclonal antibody 13A5 raised against SHa PrP did not react with the CHa or AHa PrP molecules. Comparison of the three predicted amino acid sequences revealed that each is distinct. Furthermore, differences within the PrP open reading frame that uniquely distinguish the three hamster species are within a hydrophilic segment of 11 amino acids that includes polymorphisms linked to scrapie incubation times in inbred mice and an inherited prion disease of humans. Single polymorphisms in this region correlate with the presence or absence of amyloid plaques for a given hamster species or mouse inbred strain. Our findings demonstrate distinctive molecular, pathological, and clinical characteristics of scrapie in three related species and are consistent with the hypothesis that molecular properties of the host PrP play a pivotal role in determining the incubation time and neuropathological features of scrapie.     

Two genes encode distinct glutamate decarboxylases

gamma-Aminobutyric acid (GABA) is the most widely distributed known inhibitory neurotransmitter in the vertebrate brain. GABA also serves regulatory and trophic roles in several other organs, including the pancreas. The brain contains two forms of the GABA synthetic enzyme glutamate decarboxylase (GAD), which differ in molecular size, amino acid sequence, antigenicity, cellular and subcellular location, and interaction with the GAD cofactor pyridoxal phosphate. These forms, GAD65 and GAD67, derive from two genes. The distinctive properties of the two GADs provide a substrate for understanding not only the multiple roles of GABA in the nervous system, but also the autoimmune response to GAD in insulin-dependent diabetes mellitus.     

Two distinct families of human and bovine interferon-alpha genes are coordinately expressed and encode functional polypeptides.

The classical human interferon-alpha (HuIFN-alpha) gene family is estimated to consist of 15 or more nonallelic members which encode proteins sharing greater than 77% amino acid sequence homology. Low-stringency hybridization with a HuIFN-alpha cDNA probe permitted the isolation of two distinct classes of bovine IFN-alpha genes. The first subfamily (class I) is more closely related to the known HuIFN-alpha genes than to the second subfamily (class II) of bovine IFN-alpha genes. Extensive analysis of the human genome has revealed a HuIFN-alpha gene subfamily corresponding to the class II bovine IFN-alpha genes. The class I human and bovine IFN-alpha genes encode mature IFN polypeptides of 165 to 166 amino acids, whereas the class II IFN-alpha genes encode 172 amino acid proteins. Expression in Escherichia coli of members of both gene subfamilies results in polypeptides having potent antiviral activity. In contrast to previous studies which found no evidence of class II IFN-alpha protein or mRNA expression, we demonstrate that the class I and class II IFN-alpha genes are coordinately induced in response to viral infection.     

Two homologous low-temperature-inducible genes from Arabidopsis encode highly hydrophobic proteins.

We have characterized two related cDNAs (RCI2A and RCI2B) corresponding to genes from Arabidopsis thaliana, the expression of which is transiently induced by low, nonfreezing temperatures. RCI2A and RCI2B encode small (54 amino acids), highly hydrophobic proteins that bear two potential transmembrane domains. They show similarity to proteins encoded by genes from barley (Hordeum vulgare L.) and wheatgrass (Lophophyrum elongatum) that are regulated by different stress conditions. Their high level of sequence homology (78%) and their genomic location in a single restriction fragment suggest that both genes originated as a result of a tandem duplication. However, their regulatory sequences have diverged enough to confer on them different expression patterns. Like most of the cold-inducible plant genes characterized, the expression of RCI2A and RCI2B is also promoted by abscisic acid (ABA) and dehydration but is not a general response to stress conditions, since it is not induced by salt stress or by anaerobiosis. Furthermore, low temperatures are able to induce RCI2A and RCI2B expression in ABA-deficient and -insensitive genetic backgrounds, indicating that both ABA-dependent and -independent pathways regulate the low-temperature responsiveness of these two genes.     

Alternatively spliced transcripts of the Drosophila tramtrack gene encode zinc finger proteins with distinct DNA binding specificities.

A protein present in nuclear extracts of Drosophila embryos binds multiple sites in the promoter and genetically defined autoregulatory element of the pair-rule gene even-skipped (eve). We reported here the isolation of a cDNA encoding this binding activity, the sequence of which identifies it as the 69 kDa zinc finger tramtrack (ttk) protein. As ttk was previously implicated in controlling the expression of another pair-rule gene, fushi tarazu (ftz), our findings suggest that ttk plays a role in the regulation of at least two developmentally important genes. An additional ttk-related cDNA clone was isolated which gives rise to an 88 kDa protein with an alternative set of zinc fingers having a DNA binding specificity distinct from that of the 69 kDa protein. Both proteins were shown to be encoded by the ttk gene through alternative splicing, providing the first example of the use of this mechanism to generate related proteins with distinct DNA binding specificities. Whole mount in situ hybridization analysis revealed different patterns of embryonic expression of the two ttk mRNA isoforms.     

Two functional alpha-tubulin genes of the yeast Saccharomyces cerevisiae encode divergent proteins.

Two alpha-tubulin genes from the budding yeast Saccharomyces cerevisiae were identified and cloned by cross-species DNA homology. Nucleotide sequencing studies revealed that the two genes, named TUB1 and TUB3, encoded gene products of 447 and 445 amino acids, respectively, that are highly homologous to alpha-tubulins from other species. Comparison of the sequences of the two genes revealed a 19% divergence between the nucleotide sequences and a 10% divergence between the amino acid sequences. Each gene had a single intervening sequence, located at an identical position in codon 9. Cell fractionation studies showed that both gene products were present in yeast microtubules. These two genes, along with the TUB2 beta-tubulin gene, probably encode the entire complement of tubulin in budding yeast cells.     

Two distinct isocitrate lyases from a pseudomonas species.

The isocitrate lyases of acetate- and methylamine-grown Pseudomonas MA (Shaw strain) were studied. They were shown to be different by a variety of physical criteria including chromatographic elution patterns, heat inactivation kinetics, pH variation of Km values, and migration on polyacrylamide gels. The implications and significance of the existence of two enzymes in relation to the role of isocitrate lyase in methylamine utilization is discussed.     

Three genes encode distinct AP33 proteins involved in Trichomonas vaginalis cytoadherence

Adherence to host cells is essential for the initiation and maintenance of infection by mucosal pathogens. The protozoan Trichomonas vaginalis colonizes the human urogenital tract via four surface proteins (AP65, AP51, AP33 and AP23). To characterize AP33 further, six cDNA clones were examined. Restriction mapping indicated that the six clones represented three similar genes. Southern analysis confirmed the existence of three single-copy AP33 genes and suggested a semi-conservative genomic arrangement between T. vaginalis isolates. Analysis of full-length sequences determined that each contained a 930 bp open reading frame encoding a protein of approximately 33 000 Da. Sequence comparisons revealed a high degree of identity at both the DNA and the protein levels. N-terminal protein sequencing established the presence of leader peptides. Each of the three full-length recombinant proteins had a predicted pI of approximately 10, which was verified experimentally for the T. vaginalis AP33 adhesin. A database search revealed that AP33 had significant identity to the succinyl-CoA synthetase α-subunit of several different organisms and virtually 100% identity to the reported T. vaginalis subunit. Unlike commercially purchased enzyme, the recombinant proteins retained adhesive properties equal to the natural T. vaginalis AP33. The characteristics of the AP33 protein are similar to those of the other adhesins and emphasize a complex host–parasite relationship.     

Two cyclic AMP-regulated genes from Dictyostelium discoideum encode homologous proteins

Expression of the 7E and 2C genes late in Dictyostelium development ceases upon cell disaggregation but, in contrast to many other genes we have studied, expression is fully restored by exogenous cAMP (A. J. Richards et al., submitted). The 7E and 2C genes encode polypeptides of similar size (9220 and 10573 Daltons, respectively), each of which contains an unusually high proportion of serine plus glycine residues (41% and 59%, respectively). Each protein possesses a relatively serine-rich N-terminus and glycine-rich C-terminus and contains the conserved sequence S(X)SSS(X2)SS(X)SS(X2)SFGS. These data suggest that genes 7E and 2C may have arisen by duplication of a common ancestor. Computer analysis indicates that both gene products are probably intracellular structural proteins that form extended coil structures.     

Two homologous genes, originated by duplication, encode the human hnRNP proteins A2 and A1.

Heterogeneous nuclear ribonucleoprotein (hnRNP) A2 belongs, with A1, B1 and B2, to the basic protein subset of the hnRNP complex in mammalian cells. All these proteins share a modular structure consisting of two conserved RNA binding domains linked to less conserved Gly-rich domains (2xRBD-Gly). In the framework of our studies on the genetic basis of hnRNP proteins structure and diversity we have isolated and sequenced the A2 gene and compared it to the previously described A1 gene. The A2 gene, which exists in a single copy on Ch. 7 band p15, is split in 12 exons including an alternatively spliced 36 nt mini exon specific for the human hnRNP protein B1. In this work we show that the intron/exon organisation of the A2 gene is identical to that of the A1 gene over the entire length, indicating a common origin by gene duplication. Moreover the comparison of corresponding exons evidences significant conservation also in the apparently divergent Gly-rich domains that could define previously unenvisaged structural and/or functional motifs. The A2 gene promoter is also analysed in comparison to that of the A1 gene.     

Two new lignans with their biological activities in Portulaca oleracea L.

Two new lignans, identified as 6,7-dihydroxy-4-(4′’-hydroxy-3′’-methoxyphenyl)-2-naphthoic acid, named Oleralignan C (1), and 4-(3,4-dihydroxyphenyl)-6-hydroxy-7-methoxy-2-naphthoic acid, named Oleralignan D (2), were obtained from Portulaca oleracea L. The structures were determined by spectroscopic methods, including UHPLC-ESI-QTOFMS, ¹D and ²D NMR. Both Oleralignan C (1) and Oleralignan D (2) inhibited the inflammatory factors, IL-1β and TNF-α in RAW 264.7 cells induced by lipopolysaccharide (LPS). Both compounds also could clear 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radicals indicating their antioxidant potential.