An improved automated immunoassay for C-reactive protein on the Dimension clinical chemistry system.

Recent clinical data indicate that the measurement of the concentration of C-reactive protein (CRP) requires a higher sensitivity and wider dynamic range than most of the current methods can offer. Our goal was to develop a totally automated and highly sensitive CRP assay with an extended range on the Dimension((R)) clinical chemistry system based on particle-enhanced turbidimetric-immunoassay (PETIA) technology. The improved method was optimized and compared to the Binding Site's radial immunodiffusion assay using disease state specimens to minimize interference. Assay performance was assessed on the Dimension((R)) system in a 12-instrument inter-laboratory comparison study. A split-sample comparison (n = 622) was performed between the improved CRP method on the Dimension system and the N Latex CRP mono method on the Behring Nephelometer, using a number of reagent and calibrator lots on multiple instruments. The method was also referenced to the standard material, CRM470, provided by the International Federation of Clinical Chemistry (IFCC). The improved CRP method was linear to 265.1 mg/l with a detection limit between 0.2 and 0.5mg/l. The method detects antigen excess from the upper assay limit to 2000 mg/l, thereby allowing users to retest the sample with dilution. Calibration was stable for 60 days. The within-run reproducibility (CV) was less than 5.1% and total reproducibility ranged from 1.1 to 6.7% between 3.3 and 265.4 mg/l CRP. Linear regression analysis of the results on the improved Dimension method (DM) versus the Behring Nephelometer (BN) yielded the following equation: DM = 0.99 x BN - 0.37; r = 0.992. Minimal interference was observed from sera of patients with elevated IgM, IgG and IgA. The recovery of the IFCC standard was within 100 +/- 7 % across multiple lots of reagent and calibrator. The improved CRP method provided a sensitive, accurate and rapid approach to quantify CRP in serum and plasma on the Dimension clinical chemistry system. The ability to detect antigen excess eliminated reporting falsely low results caused by the 'prozone effect'.

Transformation of microbes, plants and animals by particle bombardment.

Over the past several years, particle bombardment has evolved into a useful tool for molecular biologists, allowing direct gene transfer to a broad range of cell and tissue types. Some of the important applications of the process include the production of transgenic crop species including maize and soybean and the introduction of DNA into plastids and mitochondria. Recent results have extended the range of gene transfer by particle bombardment to animal and bacterial cells. One noteworthy newer application is the direct insertion of genes into the organs of living animals. Here we discuss these advances and the instrument developments that contributed to them.

Detection of proopiomelanocortin mRNA by in situ hybridization with an oligonucleotide probe.

Synthetic oligonucleotide probes can be easily obtained and used, in contrast to cDNA cloning to develop probes, and thus the present study was carried out to determine whether such probes could also be useful for in situ hybridization. A 24-base synthetic oligonucleotide complementary to part of the alpha-melanocyte-stimulating hormone (alpha-MSH) coding region of proopiomelanocortin (POMC) mRNA was 5'-end-labeled by using [gamma-32P]ATP with T4 polynucleotide kinase or was 3' tailed by using [alpha-32P]dATP or [3H]dCTP with terminal deoxynucleotidyltransferase. Blot analysis of pituitary poly(A)+ RNA showed that the oligonucleotide hybridized to a single species with a molecular size of approximately 1200 nucleotides, consistent with that determined previously for POMC mRNA. The oligonucleotide, regardless of labeling method, hybridized to cells in the pituitary intermediate lobe, but not in the posterior lobe. Only the 3H-labeled probe gave resolution of individual pituitary anterior lobe cells. The specificity of the hybridization was determined by showing that the intermediate lobe signal was blocked by prehybridization of the tissue with unlabeled alpha-MSH oligonucleotide probe. Furthermore, the hybridized probe exhibited a sharp sigmoid curve when melted off. Finally, the oligonucleotide probe detected, in situ, the haloperidol-induced elevation of intermediate lobe POMC mRNA. Thus, the oligonucleotide probe exhibited hybridization in an anatomically and biochemically specific manner, and it detected a tissue-specific change in mRNA levels in situ.

Glucocorticoid sensitivity of vasopressin mRNA levels in the paraventricular nucleus of the rat.

Immunocytochemical studies have shown that adrenalectomy produces changes in the content and distribution of [arginine-8]vasopressin (AVP) immunoreactivity in the paraventricular nucleus of the hypothalamus. The purpose of this study was to determine whether manipulation of adrenal hormones affects the levels of AVP mRNA. In situ hybridization assays with highly specific synthetic oligodeoxyribonucleotide probes and immunocytochemistry were used to detect the distribution of AVP mRNA and AVP-immunoreactive perikarya. AVP mRNA is codistributed with AVP immunoreactivity in the posterior magnocellular subdivision of the paraventricular nucleus and its accessory nuclei, the supraoptic nucleus and the suprachiasmatic nucleus. In adrenalectomized rats, the density and distribution of the hybridization signal were increased in the paraventricular nucleus; a 2-fold increase in the area comprising the signal was observed. At the cellular level, silver grains were detected in corticotropin-releasing-factor-immunoreactive neurons throughout the medial parvocellular subdivision of the paraventricular nucleus. No changes were seen in the distribution of AVP mRNA in the supraoptic or suprachiasmatic nuclei. Treatment with dexamethasone prevented the increase in AVP mRNA produced by adrenalectomy. In contrast, adrenalectomy did not alter the hybridization signal obtained with a probe for alpha-tubulin mRNA. These results suggest, at the cellular level, that adrenalectomy induces a glucocorticoid-sensitive stimulation of AVP mRNA synthesis in the central nervous system. Thus, considerable plasticity in gene expression is retained in the hypothalamus of the adult rat.

Rapid, high-resolution in situ hybridization histochemistry with radioiodinated synthetic oligonucleotides.

In situ hybridization histochemistry is a valuable technique for localizing specific messenger RNA (mRNA) and detecting changes in gene expression. Generally, the mRNA of interest has been detected by probes obtained from cloned DNA and labelled to high specific activity by nick translation. Such probes have a number of disadvantages which can be circumvented by the use of short synthetic oligonucleotides designed to be complementary to a known mRNA sequence. We report here that synthetic oligonucleotides complementary to part of the mRNA coding for rat arginine-vasopressin (AVP) can be labelled to high specific activity with [125I], using either the primer extension method with the Klenow fragment of DNA polymerase I or the 3'-tailing method with terminal deoxynucleotidyl transferase. Both AVP probes hybridized well to the magnocellular neurons of the hypothalamic paraventricular and supraoptic nuclei. A strong autoradiographic signal was present by 2 days, with grains largely confined to the perikaryon. These results compare favorably to those obtained with [32P]- or [3H]-labelled probes. Given the ease of the 3'-tailing method, [125I]-labelled oligonucleotides appear to be especially useful probes for in situ hybridization histochemistry.

Co-localization of corticotropin releasing factor and vasopressin mRNA in neurones after adrenalectomy.

The discrete anatomical distribution of arginine vasopressin and corticotropin releasing factor (CRF) immunoreactivity in the paraventricular nucleus (PVN) of the rat hypothalamus is altered after adrenalectomy. Not only is the immunostaining of both peptides enhanced, but vasopressin immunoreactivity, normally confined to the magnocellular subdivision, becomes clear in a large percentage of CRF neurones in the parvocellular subdivision. These changes in immunoreactivity may reflect changes in post-translational events, peptide metabolism or genomic activity that lead indirectly or directly to the enhanced expression of vasopressin. Here we report that levels of transcripts homologous to vasopressin messenger RNA increase in the PVN after adrenalectomy, in parallel with increases in vasopressin immunoreactivity. In fact, after adrenalectomy, vasopressin mRNA can be detected in CRF-immunoreactive neurones. These results indicate that a considerable degree of plasticity is retained by the adult neuronal genome of the rat and that this plasticity may be modulated by the endocrine environment.

In situ hybridization of putative somatostatin mRNA within hypothalamus of the rat using synthetic oligonucleotide probes.

The distribution of mRNA with high sequence homology to somatostatin mRNA within the periventricular hypothalamus of rat was assessed using in situ hybridization techniques with synthetic oligodeoxyribonucleotide probes, complementary to the 3' coding region of rat somatostatin mRNA. The probes (22- and 24-mers) were 5'-end labeled using T4 polynucleotide kinase and gamma-32P-ATP. They were used either individually or after ligation with T4 DNA ligase to form a 46-mer. Serial tissue sections (less than 10 microns) were taken from the level of the preoptic/anterior hypothalamus through the paraventricular hypothalamus. In situ hybridizations were conducted at room temperature in hybridization buffer. Neurons immunoreactive with antiserum raised against somatostatin were identified in alternate sections using standard immunocytochemical procedures. The anatomical location of the hybridization signal was determined by autoradiography. Our results show that the peri- and paraventricular hypothalamus is rich in transcripts putatively coding for somatostatin and that these transcripts are co-distributed with neurons immunoreactive with antisomatostatin immunoglobulin.

A site-specific mutation within the active site of ribulose-1,5-bisphosphate carboxylase of Rhodospirillum rubrum.

In vitro mutagenic techniques have generated an asp-->glu substitution at residue 198 adjacent to the carbamate-divalent metal ion binding site of Rhodospirillum rubrum ribulose 1,5-bisphosphate carboxylase. A single C-->A nucleotide change in the coding strand created the mutant and introduced a new EcoRI restriction site on the expression plasmid pRR2119. Although the carboxylase:oxygenase ratio remained the same, the mutant enzyme had slightly altered kinetic properties. The e.p.r. spectra of the quaternary complexes enzyme.activator carbamate.Mn.2-carboxyarabinitol 1,5-bisphosphate and enzyme.activator carbamate.Mn.4-carboxyarabinitol 1,5-bisphosphate for mutant and wild-type enzymes were different, indicating that the metal ion was in a slightly altered environment. These findings are consistent with the hypothesis that, besides the carbamate at lys 201, the carboxyl group of asp 198 contributes to the formation of the divalent metal ion binding site.

The sup8 tRNALeu gene of Schizosaccharomyces pombe has an unusual intervening sequence and reduced pairing in the anticodon stem.

We have cloned and sequenced the wild-type and suppressor alleles of the S. pombe sup8 tRNA gene. The wild-type allele has a leucine UAA anticodon and the suppressor (sup8-e) carries the opal suppressor anticodon UCA. The gene has a 16 base pair intervening sequence that, in the RNA, is predicted to form a secondary structure which involves base pairing to the 5', rather than the usual 3' side of the 5' splice site. When incubated in Saccharomyces cerevisiae cell-free extracts both alleles are efficiently transcribed, the 5' leader and 3' trailer sequences are removed and CCA is added to the 3' processed end; however, the intervening sequence is not excised. This finding implies that the structural requirements of the splicing endonucleases in the two yeasts have diverged. No other tRNA genes with related sequences were detected in S. pombe DNA by hybridization, suggesting that other UUA isoacceptors may be structurally dissimilar to sup8 or that the UUA codon may be decoded by a UUG leucine isoacceptor.

Introduction of restriction enzyme sites in protein-coding DNA sequences by site-specific mutagenesis not affecting the amino acid sequence: a computer program.

Structure/function relationship studies of proteins are greatly facilitated by recombinant DNA technology which allows specific amino acid mutations to be made at the DNA sequence level by site-specific mutagenesis employing synthetic oligonucleotides. This technique has been successfully used to alter one or two amino acids in a protein. Replacement of existing DNA sequence coding for several amino acids with new synthetic DNA fragments would be facilitated by the presence of unique restriction enzyme sites in the region of interest. This computer program provides a means of searching the DNA sequence of interest for restriction enzyme sites that could be introduced by site-specific mutagenesis not affecting the amino acid sequence of the protein. Alternately, the program will also allow single amino acid changes to be made.

Mechanistic study of E. coli DNA topoisomerase I: cleavage of oligonucleotides.

E. coli DNA topoisomerase I catalyzes DNA topoisomerization by transiently breaking and rejoining single DNA strands (1). When an enzyme-DNA incubation mixture is treated with alkaline or detergent, DNA strand cleavage occurs, and the enzyme becomes covalently linked to the 5'-phosphoryl end of the cleaved DNA (2). Using oligonucleotides of defined length and sequence composition, this cleavage reaction is utilized to study the mechanism of E. coli DNA topoisomerase I. dA7 is the shortest oligonucleotide tested that can be cleaved by the enzyme. dT8 is the shortest oligo(dT) that can be cleaved. The site of cleavage in both cases is four nucleotides from the 3' end of the oligonucleotide. No cleavage can be observed for oligo(dC) and oligo(dG) of length up to eleven bases long. dC15 and dC16 are cleaved at one tenth or less the efficiency of oligo(dA) and oligo(dT) of comparable length.

Thiol-beta-lactamase: replacement of the active-site serine of RTEM beta-lactamase by a cysteine residue.

We describe a procedure by which the codon (AGC) for the active-site serine-70 of pBR322 beta-lactamase (penicillinase, penicillin amido-beta-lactamhydrolase, EC 3.5.2.6) is altered to that for cysteine (TGC). The pertinent nucleotide bases, A-G-C-A, positions 410-413, of pBR322 are excised by treating a limited HgiAI digest of pBR322 with the 3' leads to 5' exonuclease of T4 DNA polymerase. The new sequence, T-G-C-A, is inserted in two steps. First, the Kpn I molecular linker d(T-G-G-T-A-C-C-A) is ligated into the gap described above. The internal sequence G-T-A-C is then excised enzymatically with Kpn I and T4 DNA polymerase and the molecule is recircularized. This mutant gene, which codes for a thiol-beta-lactamase, confers on Escherichia coli K-12 hosts an ampicillin resistance that is reduced compared with that given by pBR322 yet is greater than that of E. coli lacking any intact beta-lactamase gene. Cell-free extracts of E. coli strains hosting the thiol-beta-lactamase gene possess a p-chloromercuribenzoate-sensitive beta-lactamase activity.

Simplications in the synthesis of short oligonucleotide blocks.

A rapid and convenient procedure has been developed for the synthesis of fully protected mono, di and trideoxyribonucleotides utilizing an aryl phosphoroditriazolide. It affords advantages over coupling strategies employing condensing reagents, such as 2,4,6-triisopropylbenzenesulfonyl tetrazolide in preparing small oligonucleotides and is relatively free of the drawbacks inherent in other approaches using bifunctional phosphorylating reagents. In particular, the synthesis of trinucleotide blocks without purification at the dimer stage is described.

Solid-phase synthesis of polynucleotides. IV. Usage of polystyrene resins for the synthesis of polydeoxyribonucleotides by the phosphostriester method.

Contrary to the expectation, the Merrifield polystyrene resin, 2% cross-linked by divinylbenzene, is as efficient as the polyacrylmorpholide resin for the synthesis of polydeoxyribonucleotides using a phosphotriester method. On the Merrifield resin, the tetradecamer, dTpCpGpTpCpApApCpTpGpGpCpTpT, and the hexadecamer, dCpCpApGpTpCpApCpGpApCpGpTpTpGpT, were synthesized by the phosphotriester method using di and trinucleotide blocks as coupling units.

Direct expression in Escherichia coli of a DNA sequence coding for human growth hormone.

DNA coding for human growth hormone was constructed by using chemically synthesised DNA in conjunction with enzymatically prepared cDNA. This 'hybrid' gene was expressed in Escherichia coli under the control of the lac promoter. A polypeptide was produced having the size and immunological properties characteristic of mature human growth hormone.