Assessment of a molecular diagnostic platform for integrated isolation and quantification of mRNA in whole blood.

Implementation of point-of-care tests may facilitate the health management of infectious diseases by reducing the timeframe on pathogen identification and host response measurements, allowing for immediate diagnosis and guided clinical intervention. In this feasibility study, a novel totally integrated and fully automated real-time polymerase chain reaction (PCR) platform (Idylla™, Biocartis) was assessed to determine the mRNA expression levels of multiple genes from 1 mL of whole blood. To this purpose, a sample-in result-out assay, including mRNA extraction and RT-qPCR-based detection, was ported to the platform. The genes used (matrix metallopeptidase 9, olfactomedin 4, NB1 glycoprotein and lipocalin 2) were previously identified as predictive for severity of disease caused by infection with respiratory syncytial virus (RSV). The reproducibility and robustness of the prototype assay was determined using the blood samples of 21 healthy donors. The data showed that the Idylla™ platform allows for a fast and user-friendly determination of the relative expression levels of the four selected mRNA markers.

The Saccharomyces cerevisiae phosphotyrosyl phosphatase activator proteins are required for a subset of the functions disrupted by protein phosphatase 2A mutations.

In Saccharomyces cerevisiae, PTPA is encoded by two genes, YPA1 and YPA2. In order to examine the biological role of PTPA as potential regulator of protein phosphatase 2A (PP2A), we compared the phenotypes of the ypaDelta mutants with these of PP2A-deficient strains. While deletion of both YPA genes is lethal, deletion of YPA1 alone results in a phenotype resembling that of PP2A-deficient strains in specific aspects such as aberrant bud morphology, abnormal actin distribution, and similar growth defects under various growth conditions. These phenotypes were even more pronounced when YPA1 was deleted in a pph21Delta genetic background. Moreover, ypaDelta mutants are hypersensitive to nocodazole and show inappropriate mitotic spindle formation as previously described for mutants in the catalytic subunit of PP2A, suggesting that Ypa, like PP2A, has a function in mitotic spindle formation. These results are consistent with an in vivo role of Ypa as a regulator of PP2A. However, unlike a PP2A-deficient strain, ypaDelta mutants do not show a G2 arrest. Therefore, Ypa does not seem to play a role in the regulation of PP2A at this stage of the cell cycle. These results imply that Ypa regulates a specific subset of PP2A functions, possibly by controlling the subunit composition of PP2A.

The Saccharomyces cerevisiae homologue YPA1 of the mammalian phosphotyrosyl phosphatase activator of protein phosphatase 2A controls progression through the G1 phase of the yeast cell cycle.

The Saccharomyces cerevisiae gene YPA1 encodes a protein homologous to the phosphotyrosyl phosphatase activator, PTPA, of the mammalian protein phosphatase type 2A (PP2A). In order to examine the biological role of PTPA, we disrupted YPA1 and characterised the phenotype of the ypa1Delta mutant. Comparison of the growth rate of the wild-type strain and the ypa1Delta mutant on glucose-rich medium after nutrient depletion showed that the ypa1Delta mutant traversed the lag period more rapidly. This accelerated progression through "Start" was also observed after release from alpha-factor-induced G1 arrest as evidenced by a higher number of budding cells, a faster increase in CLN2 mRNA expression and a more rapid reactivation of Cdc28 kinase activity. This phenotype was specific for deletion of YPA1 since it was not observed when YPA2, the second PTPA gene in budding yeast was deleted. Reintroduction of YPA1 or the human PTPA cDNA in the ypa1Delta mutant suppressed this phenotype as opposed to overexpression of YPA2. Disruption of both YPA genes is lethal, since sporulation of heterozygous diploids resulted in at most three viable spores, none of them with a ypa1Delta ypa2Delta genotype. This observation indicates that YPA1 and YPA2 share some essential functions. We compared the ypa1Delta mutant phenotype with a PP2A double deletion mutant and a PP2A temperature-sensitive mutant. The PP2A-deficient yeast strain also showed accelerated progression through the G1 phase. In addition, both PP2A and ypa1Delta mutants show similar aberrant bud morphology. This would support the notion that YPA1 may act as a positive regulator of PP2A in vivo.

Identification and characterization of alternative splice products encoded by the human phosphotyrosyl phosphatase activator gene.

The phosphotyrosyl phosphatase activator (PTPA), a protein phosphatase 2A (PP2A) regulatory protein, specifically stimulates the phosphotyrosyl phosphatase activity of PP2A in vitro. Human PTPA is encoded by a single gene, the structure and chromosomal localization of which have been determined in our previous work. In this paper, we report the identification and characterization of six additional splice variants, termed PTPAbeta to PTPAeta, in addition to the originally identified PTPAalpha form. Interestingly, PTPAbeta and PTPAgamma contain a novel exon that had been overlooked in the formerly identified gene structure. As revealed by nested PCR, all these PTPA transcripts are expressed in various human cDNA libraries and cell lines. However, a quantitative approach, using a single PCR reaction followed by detection of the reaction products with a radioactively labeled probe, revealed only PTPAalpha, beta and delta, suggesting that the other transcripts are expressed very poorly. In vitro transcription-translation revealed that only PTPAalpha, beta, delta and epsilon are translated into functional proteins, whereas translation of PTPAgamma, zeta and eta is stopped prematurely due to a frameshift resulting from skipping exon 2, suggesting that the latter isoforms may result from splicing errors. By western analysis of HepG2 and Saos-2 cell extracts, only PTPAalpha and beta were detected. PTPAalpha and beta were expressed as GST fusion proteins in bacteria, and were found to contain the same specific phosphotyrosyl phosphatase stimulatory activity towards PP2A. The identification of this family of PTPA variants adds another level of complexity to the in vivo function(s) of PTPA, opening up the possibility that different isoforms may perform different functions.

The phosphotyrosyl phosphatase activator gene is a novel p53 target gene.

The minimal promoter of the phosphotyrosyl phosphatase activator (PTPA) gene, encoding a regulator of protein phosphatase 2A contains two yin-yang 1 (YY1)-binding sites, positively regulating promoter activity. We now describe a role for p53 in the regulation of PTPA expression. Luciferase reporter assays in Saos-2 cells revealed that p53 could down-regulate PTPA promoter activity in a dose-dependent manner, whereas four different p53 mutants could not. The p53-responsive region mapped to the minimal promoter. Overexpression of YY1 reverses the repressive effect of p53, suggesting a functional antagonism between p53 and YY1. The latter does not involve competition for YY1 binding, but rather direct control of YY1 function. Inhibition of PTPA expression by endogenous p53 was demonstrated in UVB-irradiated HepG2 cells, both on the mRNA and protein level. Also basal PTPA levels are higher in p53-negative (Saos-2) versus p53-positive (HepG2, U2OS) cells, suggesting "latent" p53 can control PTPA expression as well. The higher PTPA levels in U2OS cells, programmed to overexpress constitutively a dominant-negative p53 mutant, corroborate this finding. Thus, PTPA expression is negatively regulated by p53 in normal conditions and in conditions where p53 is up-regulated, via an as yet unknown mechanism involving the negative control of YY1.

Functional analysis of the promoter region of the human phosphotyrosine phosphatase activator gene: Yin Yang 1 is essential for core promoter activity.

The phosphotyrosine phosphatase activator (PTPA) has been isolated as an in vitro regulator of protein phosphatase 2A. Human PTPA is encoded by a single gene, the structure and chromosomal localization of which have been determined in our previous work. Here we describe the further isolation, sequencing and functional characterization of the PTPA promoter region. In agreement with its ubiquitous expression, the PTPA promoter displays several characteristics of housekeeping genes: it lacks both a TATA-box and a CAAT-box, it is very GC-rich and it contains an unmethylated CpG island surrounding the transcription initiation site. Transient transfection experiments in different cell types with several truncated chimaeric luciferase reporter gene plasmids revealed the importance of the region between positions -67 and -39 for basal promoter activity. This region coincides remarkably well with the determined CpG island. Further analysis of this region demonstrated the presence of a Yin Yang 1 (YY1) binding motif at positions -52 to -44. Binding of YY1 to this sequence is demonstrated in bandshift and DNase I footprinting experiments. Another YY1 binding motif is found in the 5' untranslated region, at positions +27 to +35. Mutations in either of these sites, abolishing YY1 binding in vitro, have differential effects on promoter activity. Point mutations in both sites completely abolish promoter activity. Moreover, induction of promoter activity by co-transfection with a YY1 expression plasmid is fully dependent upon the presence of both intact YY1 binding sites. Thus YY1 apparently mediates basal transcription of the human PTPA gene through two binding sites within its proximal promoter.

Purification of porcine brain protein phosphatase 2A leucine carboxyl methyltransferase and cloning of the human homologue.

The carboxyl methyltransferase, which is claimed to exclusively methylate the carboxyl group of the C-terminal leucine residue of the catalytic subunit of protein phosphatase 2A (Leu(309)), was purified from porcine brain. On the basis of tryptic peptides, the cDNA encoding the human homologue was cloned. The cDNA of this gene encodes for a protein of 334 amino acids with a calculated M(r) of 38 305 and a predicted pI of 5.72. Database screening reveals the presence of this protein in diverse phyla. Sequence analysis shows that the novel methyltransferase is distinct from other known protein methyltransferases, sharing only sequence motifs supposedly involved in the binding of adenosylmethionine. The recombinant protein expressed in bacteria is soluble and the biophysical, catalytic, and immunological properties are indistinguishable from the native enzyme. The methylation of PP2A by the recombinant protein is restricted to Leu(309) of PP2A(C). No direct effects on phosphatase activity changes were observed upon methylation of the dimeric or trimeric forms of PP2A.

Transposase is the only nematode protein required for in vitro transposition of Tc1.

The Tc1 element of Caenorhabditis elegans is a member of the most widespread class of DNA transposons known in nature. Here, we describe efficient and precise transposition of Tc1 in a cell-free system. Tc1 appears to jump by a cut-and-paste mechanism of transposition. The terminal 26 bp of the Tc1 terminal repeats together with the flanking TA sequence are sufficient for transposition. The target site choice in vitro is similar to that in vivo. Transposition is achieved with an extract prepared from nuclei of transgenic nematodes that overexpress Tc1 transposase but also by recombinant transposase purified from Escherichia coli. The simple reaction requirements explain why horizontal spread of Tc1/mariner transposons can occur. They also suggest that Tcl may be a good vector for transgenesis of diverse animal species.

Formation of two hydrogen bonds from the globin to the heme-linked oxygen molecule in Ascaris hemoglobin.

We have tried to find out why Ascaris hemoglobin has such an exceptionally high oxygen affinity (P50 approximately 0.004 mmHg; 1 mmHg = 133 Pa). Following Kloek et al., we have synthesized the N-terminal globin domain of Ascaris hemoglobin in Escherichia coli [Kloek, A. P., Yang, J., Mathews, F. S. & Goldberg, D. (1993) J. Biol. Chem. 268, 17669-17671]. Like Kloek et al., we found its oxygen affinity to be as high as that of native Ascaris hemoglobin. We thought that this high affinity might be due to the heme-bound oxygen molecule being stabilized by two hydrogen bonds from the globin instead of the usual one. Ascaris hemoglobin has a distal glutamine instead of the more usual histidine as one of the potential hydrogen bond donors. In addition, it contains a tyrosine at position 10 of B helix (B10) in place of the leucine generally found there in vertebrate myoglobins and hemoglobins. Following the discovery of Carver et al. that sperm whale myoglobin with the replacement of leucine B10 by phenylalanine has a raised oxygen affinity, we have replaced tyrosine B10 in the N-terminal domain of Ascaris hemoglobin by either leucine or phenylalanine [Carver, T. E., Brantley, R. E., Jr., Singleton, E. W., Arduini, R. M., Quillin, H. L., Phillips, G. N., Jr., & Olson, J. S. (1992) J. Biol. Chem. 267, 14443-14450]. Either of these replacements lowered the oxygen affinity about 100-fold, to the same level of that of human alpha-globin chains. These results are consistent with a hydrogen bond linking the tyrosine hydroxyl to the heme-linked oxygen, with a bond energy of 2.7 kcal/mol.

Polar zippers.

BACKGROUND: Certain proteins are known to form leucine zippers - alpha-helical coiled-coils in which the non-polar side chains of two leucine-rich helices intermesh. We recently presented the first evidence for a polar zipper, formed by the carboxy-terminal peptides of the eight subunits of Ascaris haemoglobin. The evidence was based on the presence of pairs of acidic residues alternating with pairs of basic residues ( + + - - ) in an amino-acid sequence that has since been shown to be incomplete. The complete sequence, derived from the haemoglobin's cDNA, now shows a self-complementary polar sequence extending along the entire length of its 24-residue carboxy-terminal peptide. RESULTS: From the complete sequence, it is clear that the eight identical subunits of the haemoglobin could be held together by an eight-stranded antiparallel beta barrel made up of the carboxy-terminal 24 residues of each of the subunits, such that each strand forms 10 salt bridges with each of its neighbours. A computer search of the protein database revealed similar, but shorter, + + - - repeats in several other proteins. It also revealed long repeats of alternating arginine and aspartate residues, and long stretches of only glutamines, or only serines, suggestive of several other kinds of polar zippers. CONCLUSION: Several proteins have amino-acid sequences that suggest the formation of polar zippers made of beta strands. These could form antiparallel pleated sheets linked together by hydrogen bonds between polar side chains both above and below the plane of the sheets. Polar zippers may be important in welding together oligomeric proteins which have subunits lacking the extensive complementary surfaces necessary for stability, or in promoting the association of functionally complementary proteins.

Molecular characterization of an extracellular acid-resistant lipase produced by Rhizopus javanicus.

An extracellular lipase (triacylglycerol acylhydrolase EC 3.1.1.3), produced by the fungus Rhizopus javanicus was purified to homogeneity using an expeditious two-step isolation method. The enzyme, with a molecular mass of 36 kDa and a specific activity of 9260 microequivalent of fatty acid released per minute and mg under standard conditions, consists of three isoforms with isoelectric points of 7.8, 7.7, and 7.1, respectively. The purified lipase was digested using chemical and enzymatical procedures: CNBr cleavage, partial acid hydrolysis, and proteolytic cleavage by means of trypsin. Amino-acid sequencing of the resulting peptides indicates that the three lipases from Rhizopus javanicus, Rhizopus niveus and Rhizopus delemar are produced as identical proenzymes but processed differently. These Rhizopus lipases show 54% identity with the lipase from Rhizomucor miehei. Using the structure of the Rhizomucor miehei lipase, the molecular model of Rhizopus javanicus lipase was constructed. Both enzymes are alpha/beta type proteins with a central 8-stranded mixed beta-pleated sheet and have a remarkably similar distribution of hydrophobic amino acids at their surface. The tryptophan in the center of the helical lid covering the active site of Rhizomucor miehei lipase is mutated into an alanine, indicating that it is not essential for the proper movement of the helical lid.

Unexpected intron location in non-vertebrate globin genes.

The Caenorhabditis elegans and Artemia T4 globin sequences are highly homologous with other invertebrate globins. The intron/exon patterns of their genes display a single intron in the E and G helices respectively. Precoding introns in multirepeat globins are inserted in homologous positions. Comparison of the intron/exon patterns in the known globin gene sequences demonstrates that they are more diverse than first expected but nevertheless can be derived from an ancestral pattern having 3 introns and 4 exons.

Polar zipper sequence in the high-affinity hemoglobin of Ascaris suum: amino acid sequence and structural interpretation.

The extracellular hemoglobin of Ascaris has an extremely high oxygen affinity (P50 = 0.004 mmHg). It consists of eight identical subunits of molecular weight 40,600. Their sequence, determined by protein chemistry, shows two tandemly linked globin-like sequences and an 18-residue C-terminal extension. Two N-linked glycosylation sites contain equal ratios of mannose/glucosamine/fucose of 3:2:1. Electron micrographs suggest that the eight subunits form a polyhedron of point symmetry D4, or 42. The C-terminal extension contains a repeat of the sequence Glu-Glu-His-Lys, which would form a pattern of alternate glutamate and histidine side chains on one side and of glutamate and lysine side chains on the other side of a beta strand. We propose that this represents a polar zipper sequence and that the C-terminal extensions are joined in an eight-stranded beta barrel at the center of the molecule, with histidine and glutamate side chains inside and lysine and glutamate side chains outside the barrel compensating each other's charges. The amino acid sequence of Ascaris hemoglobin fails to explain its high oxygen affinity.

The primary structure of a minor isoform (H1.2) of histone H1 from the nematode Caenorhabditis elegans.

The complete amino acid sequence of a minor isoform (H1.2) of histone H1 from the nematode Caenorhabditis elegans was determined. The amino acid chain consists of 190 residues and has a blocked N-terminus. Histone subtype H1.2 is 17 residues shorter than the major isoform H1.1, mainly as the result of deletions of short peptide fragments. Considerable divergence from isoform H1.1 has occurred in the N-terminal domain and the very C-terminus of the molecule, but the central globular domain and most of the C-terminal domain, including two potential phosphorylation sites, have been well conserved. Secondary-structure predictions for both H1 isoforms reveal a high potential for helix formation in the N-terminal region 1-33 of isoform H1.1 whereas the corresponding region in isoform H1.2 has low probability of being found in alpha-helix. No major differences in secondary structure are predicted for other parts of both H1 subtypes. The aberrant conformation of isoform H1.2 may be indicative of a significantly different function.