Biologically-generated primer for PCR: PCR primer of unknown sequence.

We describe a method for producing specific PCR primers directly from PCR product, bypassing the usual need to know the primer sequence. Lack of abundance of primers derived from a PCR product is compensated for by the incorporation of an arbitrary 5'TAG sequence which acts as a surrogate template target for the bulk amplification phase. We use the technique to amplify clonospecific rearranged immunoglobulin genes, which have applications as markers of lymphoid neoplasms for tracing the success of therapy. The principle may have wider application wherever conserved and variable regions of DNA are juxtaposed.     

Segmental specificity and lateral asymmetry in the differentiation of developmentally homologous neurons during leech embryogenesis

This paper describes the embryonic development of three leech neurons which undergo spatially regulated patterns of differentiation. In leeches, the nervous system arises from an iterated array of embryonic cell lineages, and each neuron is represented by a set of bilaterally symmetric and segmentally repeated homologs. Two of the cells discussed here, the neurons nz4 and mz3, stain with antibodies to the neuropeptides SCP and FMRFamide during the course of their embryonic differentiation, but only a subset of the initially immunoreactive homologs continue to express this immunoreactivity into postembryonic life. Those nz4 cells which retain immunoreactivity are referred to as RAS neurons, and the persistently immunoreactive mz3 cells referred to as CAS neurons. The subset of homologs which show persistent expression is segment specific, such that the mature RAS and CAS neurons occupy different segmental domains. In addition, both neurons display a final pattern of expression which is laterally asymmetric, with only one of the two homologs in each segment maintaining the RAS or CAS phenotype. Asymmetric differentiation can occur in either orientation for any given segment, although there is a very strong tendency for the persistently immunoreactive cells to lie on opposite sides of successive segments. The fate of the transiently immunoreactive homologs is unclear, but labeling with intracellular lineage tracers suggests that there are some mz3 neurons which survive late into postemobryonic life and never express detectable levels of immunoreactivity. Intracellular lineage tracers also allowed us to follow the development of a third neuron, mz4, which does not stain for either peptide. The mz4 neuron is initially paired, but undergoes an asymmetric pattern of cell death which also shows a strong tendency to alternate sides in successive segments. These spatially coordinated patterns of neuronal survival and/or differentiation suggest that cell interactions play a role in determining the developmental choices made by individual neurons, and a subsequent paper will characterize those interactions through experimental manipulation.     

Isolation and complete sequence of the purL gene encoding FGAM synthase II in Lactobacillus casei.

The purL gene from Lactobacillus casei, encoding phosphoribosylformylglycinamidine synthase II involved in the de novo synthesis of purines, was cloned and sequenced. The putative purL product of 741 amino acids (M(r) of 79,575) shows 25% and 53% identity to the homologous enzymes from Escherichia coli and Bacillus subtilis, respectively. In addition, partial sequences of two other pur genes (purQ and purF) and a possible third gene (purC) were obtained. All these genes are organized in an operon similar to that of B. subtilis. In contrast, the corresponding genes from E. coli and Salmonella typhimurium are scattered through the genome.     

Pulmonary aspiration in preschool children with cystic fibrosis

Pulmonary aspiration of gastric refluxate (PAGR) has been demonstrated in association with pulmonary inflammation in school aged children with Cystic Fibrosis (CF). We sought to determine if similar findings were present in preschool children. Pepsin was measured in Broncho-alveolar lavage (BAL) fluid collected from clinically stable preschool children with CF and controls. Elevated pepsin levels were found in a subgroup of children with CF, but this was not found to be associated with pulmonary infection, pulmonary inflammation or respiratory or gastrointestinal symptoms.     

Acclimation of photosynthesis to low temperature in Spinacia oleracea L. II. Effects of nitrogen supply

The photosynthetic capacity of leaves of N-sufficent plantsof Spinacia oleracea L. increases following transfer a constanttemperature of 10C for 10 d compared to plants maintained at25C. The effects of nitrogen nutrition on this low temperatureacclimation have been investigated in respect of CO₂ assimilation,the activities and activation states of key enzymes and thepartitioning of recently fixed carbon. N-deficiency greatlyrestricted acclimation of photosynthetic CO₂ assimilation tolow temperature at both ambient and at saturating CO₂ concentrations,indicating a restriction on accilmatory changes in both ribulose1,5-bisphosphatecarboxylase-oxygenase (Rubisco) and the reactions of ribulose1,5-bisphosphateregeneration. Nitrogen limitation led to an increase in thepartitioning of recently-fixed carbon into starch. Total proteinincreased during acclimation in both N-sufficient and N-deficientleaves and was much less affected than were the activities ofenzymes. Increases in the activation state of Rubisco and thestromal fructose-1,6-bisphosphatase occurred in response tolow temperature, but increases in the activities of Rubisco,sucrose-phosphate synthase or the cytosolic fructose1,6-bisphosphatasecould not be sustained in N-deficient plants throughout theperiod of acclimation, although the activities of these enzymesdeclined less precipitately than in non-acclimated N-deficientplants. These data are all consistent with the view that increasesin the activities of key enzymes of carbon assimilation area pre-requisite for acclimation to low temperature and thatthese increases are restricted under N-limitation. Key words: Low temperature, nitrogen, photosynthesis, Rubisco, sucrose-phosphate synthase     

Dextran sulfate can act as an artificial receptor to mediate a type-specific herpes simplex virus infection via glycoprotein B.

Herpes simplex virus (HSV) adsorption to host cells is mediated, at least in part, by the interaction of viral glycoproteins with cell surface glycosaminoglycans such as heparan sulfate and chondroitin sulfate. To investigate the contribution of various cell surface components in the infection pathway, we isolated a mutant cell line, sog9, which is unable to synthesize glycosaminoglycans (B. W. Banfield, Y. Leduc, L. Esford, K. Schubert, and F. Tufaro, J. Virol. 69:3290-3298, 1995). Although HSV-1 and HSV-2 infection of sog9 cells is diminished, the cells are still infected at about 0.5% efficiency, which suggests that these cells normally express at least one nonglycosaminoglycan receptor. In this report, we used sog9 cells to test whether glycosaminoglycan analogs, such as dextran sulfate (DS), could functionally substitute for cellular glycosaminoglycans to initiate HSV infection. We show that high-molecular-weight DS added either prior to or during inoculation stimulated HSV-1 but not HSV-2 infection by up to 35-fold; DS added after viral adsorption had no effect on infection efficiency. Moreover, DS stimulated HSV-1 infection at 4 degrees C, indicating that this compound impinged on an early, energy-independent step in infection. Using radiolabeled virus, we showed that HSV-1 is more efficient than HSV-2 in adsorbing to DS immobilized on microtiter wells. This raised the possibility that only HSV-1 could engage additional receptors to initiate infection in the presence of DS. To determine which viral component(s) facilitated DS stimulation, a panel of intertypic recombinants and deletion mutant viruses was investigated. These assays showed that DS stimulation of infection is mediated primarily by gB-1. Thus, this study provides direct evidence that a principal role for cell surface glycosaminoglycans in HSV infection is to provide an efficient matrix for virus adsorption. Moreover, by using DS as an alternative adsorption matrix (a trans receptor), we uncovered a functional, type-specific interaction of HSV-1 with a cell surface receptor.     

Multiple introns in a conjugation-specific gene from Tetrahymena thermophila

Multiple introns have been found in a gene from a ciliated protozoan. This Tetrahymena thermophila gene (cnjB) is large (7.5 kb mRNA) and active only during conjugation, the organism's sexual cycle. Six introns ranging in size from 62 bp to 676 bp were found when we sequenced a 3.1 kb segment of the cnjB gene together with its corresponding cDNA. We estimate, by extrapolation of our current data, a total of approximately 30 introns in this gene with a total gene size (introns plus exons) of 15 kb or more. The number of introns is surprising given the scarcity of introns in ciliate genes examined to date. Our findings constitute the first example of multiple introns in a ciliate gene. Having the sequence of several introns has allowed us to construct consensus sequences for T. thermophila mRNA introns. The 5' and 3' intron junctions resemble those of general nuclear mRNA (GT/AG rule is followed) but differences are seen. In particular, stretches of 10 or more adenines and thymines are found adjacent to the conserved GT and AGs at the junctions. Unusual aspects of the coding region of this gene are discussed.     

Bi‐Functional Iron‐Only Electrodes for Efficient Water Splitting with Enhanced Stability through In Situ Electrochemical Regeneration

Scalable and robust electrocatalysts are required for the implementation of water splitting technologies as a globally applicable means of producing affordable renewable hydrogen. It is demonstrated that iron‐only electrode materials prove to be active for catalyzing both proton reduction and water oxidation in alkaline electrolyte solution with superior activity to that of previously established bi‐functional catalysts containing less abundant elements. The reported bi‐functionality of the iron electrodes is reversible upon switching of the applied bias through electrochemical interconversion of catalytic species at the electrode surface. Cycling of the applied bias results in in‐situ electrochemical regeneration of the catalytic surfaces and thereby extends the catalyst stability and lifetime of the water electrolyzer. Full water splitting at a current density of I = 10 mA cm^?2 is achieved at a bias of ≈2 V, which is stable over at least 3 d (72 one hour switching cycles). Thus, potential‐switching is established as a possible strategy of stabilizing electrode materials against degradation in symmetrical water splitting systems.