Two histone deacetylases, FfHda1 and FfHda2, are important for Fusarium fujikuroi secondary metabolism and virulence.

Histone modifications are crucial for the regulation of secondary metabolism in various filamentous fungi. Here we studied the involvement of histone deacetylases (HDACs) in secondary metabolism in the phytopathogenic fungus Fusarium fujikuroi, a known producer of several secondary metabolites, including phytohormones, pigments, and mycotoxins. Deletion of three Zn(2+)-dependent HDAC-encoding genes, ffhda1, ffhda2, and ffhda4, indicated that FfHda1 and FfHda2 regulate secondary metabolism, whereas FfHda4 is involved in developmental processes but is dispensable for secondary-metabolite production in F. fujikuroi. Single deletions of ffhda1 and ffhda2 resulted not only in an increase or decrease but also in derepression of metabolite biosynthesis under normally repressing conditions. Moreover, double deletion of both the ffhda1 and ffhda2 genes showed additive but also distinct phenotypes with regard to secondary-metabolite biosynthesis, and both genes are required for gibberellic acid (GA)-induced bakanae disease on the preferred host plant rice, as Δffhda1 Δffhda2 mutants resemble the uninfected control plant. Microarray analysis with a Δffhda1 mutant that has lost the major HDAC revealed differential expression of secondary-metabolite gene clusters, which was subsequently verified by a combination of chemical and biological approaches. These results indicate that HDACs are involved not only in gene silencing but also in the activation of some genes. Chromatin immunoprecipitation with the Δffhda1 mutant revealed significant alterations in the acetylation state of secondary-metabolite gene clusters compared to the wild type, thereby providing insights into the regulatory mechanism at the chromatin level. Altogether, manipulation of HDAC-encoding genes constitutes a powerful tool to control secondary metabolism in filamentous fungi.

Long-range structure in ribonuclease P RNA.

Phylogenetic-comparative and mutational analyses were used to elucidate the structure of the catalytically active RNA component of eubacterial ribonuclease P (RNase P). In addition to the refinement and extension of known structural elements, the analyses revealed a long-range interaction that results in a second pseudoknot in the RNA. This feature strongly constrains the three-dimensional structure of RNase P RNA near the active site. Some RNase P RNAs lack this structure but contain a unique, possibly compensating, structural domain. This suggests that different RNA structures located at different positions in the sequence may have equivalent architectural functions in RNase P RNA.

Molecular cloning of sheep lung dipeptidase: a glycosyl phosphatidylinositol-anchored ectoenzyme that converts leukotriene D4 to leukotriene E4.

Sheep lung dipeptidase was released from a plasma membrane preparation by digestion with a phosphatidylinositol-specific phospholipase C. The dipeptidase was purified to homogeneity using affinity chromatography followed by high performance liquid chromatography. The NH2-terminal sequence was determined and employed to prepare a probe to clone the c-DNA of the enzyme. The primary structure of sheep lung dipeptidase, deduced from the c-DNA exhibited a high homology to kidney dipeptidases cloned from other animal species. Northern analysis detected the m-RNA of the dipeptidase in lung, kidney, and intestinal tissues of the sheep.

Suppression of loss-of-function mutations in Escherichia coli ribonuclease P RNA (M1 RNA) by a specific base-pair disruption.

A plasmid encoding ribonuclease P RNA of Escherichia coli (M1 RNA) was mutagenized with hydroxylamine in vitro and defective rnpB genes were identified by screening in an in vivo suppression assay. Defective rnpB sequences were mutagenized with a second round of hydroxylamine to restore activity. We report here that conversion of the C32.G48 base-pair of RNase P RNA to either C.A or U.G restored activity to defective rnpB genes bearing a variety of spatially distinct primary mutations. Disruption of this base-pair in an otherwise wild-type rnpB sequence increased the growth rate of the indicator strain E. coli FS101, consistent with the opening of C32.G48 during in vivo assembly of or catalysis by RNase P.

Glucose concentration in subcutaneous extracellular space.

OBJECTIVE: To compare the subcutaneous glucose sensor measurements with two reference methods. Previous studies provide conflicting findings about the real glucose concentrations in subcutaneous tissue. Some suggest substantially lower concentration, whereas others measure proportionally higher glucose concentrations compared with the blood compartment. Before these results can be taken seriously as an expression of the real glucose concentration in the extracellular space, the measurements must be validated by an independent method. RESEARCH DESIGN AND METHODS: We applied a microdialysis-based enzyme sensor to measure glucose concentration in subcutaneous tissue. We also developed two reference methods: subcutaneous filtrate collection and an equilibration method using ultrafiltration membranes to support the earlier findings. We provided an anatomical model to explain the results. RESULTS: The mean overall intercellular filtrate glucose concentration, sampled with the filtrate collector and taken after a 6-h stabilization time, including the values during the glucose clamp period, was 46 +/- 9%. The mean subcutaneous glucose concentration measured with the glucose sensor, calibrated in vitro, was 44 +/- 8% of the mean venous blood glucose concentration. Mean overall intercellular equilibrate glucose concentration, i.e., the mean glucose concentration in the subcutaneous extracellular space, taken after a 4-h stabilization time, was 46 +/- 15% of the mean venous blood glucose concentration. CONCLUSIONS: The close agreement between the mean values of subcutaneous glucose concentrations, obtained with three independent methods--filtration, equilibration, and dialysis (sensor)--shows the real glucose concentration in subcutaneous interstitial fluid is approximately 50% the blood glucose value in normal humans. Our results clarify some of the conflicting evidence presented in previous studies.

Sequences encoding the protein and RNA components of ribonuclease P from Streptomyces bikiniensis var. zorbonensis.

The genes encoding the RNA (rnpB) and protein (rnpA) subunits of ribonuclease P (RNase P) of Streptomyces bikiniensis var. zorbonensis have been cloned by complementing the temperature-sensitive growth phenotype of Escherichia coli strains that carry mutations in these genes. The rnpB sequence of S. bikiniensis includes new covariations that lead to refinement of the previous secondary structure models for RNase P RNAs. The deduced amino acid sequence of S. bikiniensis RNase P is conserved with that of other known RNase P proteins only to a limited extent. Immediately upstream from rnpA is an open reading frame that codes for the highly conserved ribosomal protein, L34. This same gene arrangement occurs in all bacteria studied to date.

Gene organization in the bleomycin-resistance region of the producer organism Streptomyces verticillus.

A nucleotide sequence of 7 kb is reported, encompassing two bleomycin-resistance (BmR-encoding) genes and five other open reading frames (ORFs) from the Bm-producing organism Streptomyces verticillus ATCC 15003. The deduced ORFs, in sequence order, encode for (i) a protein homologous to an amino-acid dioxygenase; (ii) BlmA, the BmR-binding protein described by Sugiyama et al. [Gene 151 (1994) 11-16]; (iii) a product containing three copies of a sequence homologous to the ankyrin repeat; (iv) a product lacking homology to any of the sequences in the Protein Identification Resource database (PIR), release 37; (v) BlmB, the BmR acetyltransferase described by Sugiyama et al. (1994); (vi) an unidentified protein which augmented resistance determined by ORF2 (BlmA); (vii) a member of the ATP-binding cassette (ABC) family of transport protein. Predicted translational frameshifts in the -1 frame occur at the junctions between ORF3 and ORF4, ORF4 and ORF5, and ORF6 and ORF7. Sequences homologous to ORF2 and ORF3 were identified in the genome of the producer organism for the related antibiotic phleomycin.

Transcript hairpin structures are not required for RNA polymerase pausing in the gene encoding the E. coli RNase P RNA, M1 RNA.

Strong pauses at nucleotides +118 and +121 relative to the transcriptional start occur during in vitro transcription of the E. coli rnpB gene encoding the catalytic M1 RNA subunit of Ribonuclease P. These pauses are immediately downstream of 2 phylogenetically conserved stem-loop structures in the RNA. In the present work, single-base changes which disrupted Watson-Crick base-pairing in the hairpins were introduced into rnpB. Transcription studies in vitro with these modified templates revealed that none of the nucleotide changes predicted to increase or decrease the stability of the first hairpin significantly affected the pause half-lives. A mutation which disrupted the second hairpin increased the pause half-life 2-fold. The data suggest that the upstream stem and loop structures in the transcript are not involved in the pausing event.

In vitro transactivation of Bacillus subtilis RNase P RNA.

Deletion of the 'signature' PL5.1 stem-loop structure of a Type II RNase P RNA diminished its catalytic activity. Addition of PL5.1 in trans increased catalytic efficiency (kcat/KM) rather than kcat. Transactivation was due to the binding of a single PL5.1 species per ribozyme with an apparent Kd near 600 nM. The results are consistent with the role of PL5.1 being to position the substrate near the active site of the ribozyme, and with the hypothesis that ribozymes can evolve by accretion of preformed smaller structures.

RNA libraries and RNA recognition.

Random RNA libraries, consisting of > 10(13) unique sequences, contain molecules capable of specifically binding small molecule and protein ligands by noncovalent interaction. Successive steps of affinity purification and amplification allow the propagation and eventual isolation of specific binding molecules, called aptamers. Although protein- and small-molecule-binding aptamers have been characterized previously, selection of RNA aptamers capable of binding nucleic acids has previously yielded only molecules capable of Watson-Crick base pairing to the nucleic acid ligand used for selection. On the other hand, it is known from studies on catalytic and other RNAs that both inter- and intramolecular RNA-RNA interaction can occur by non-Watson-Crick means. We have therefore incorporated a strategy to obviate the possibility of Watson-Crick interaction into a selection scheme for the isolation of RNA-recognizing aptamers. The aptamers so isolated do not show extensive Watson-Crick complementarity with the RNA ligand used for the selection, thereby validating the selection strategy. Curiously, all of the aptamers characterized contain several oligo-G stretches bounded by U residues. This sequence motif, which occurs as DNA in telomeres (chromosome ends) may therefore be a general RNA-RNA interaction motif. An additional sequence motif is apparently superimposed on this background structure.

Ribozymes--why so many, why so few?

The RNA world scenario posits the existence of catalytic and genetic networks whose reactions are catalyzed by RNAs. Substantial progress has been made in recent years in the selection of RNA catalysts by SELEX, thus verifying one prediction of the model. However, many selected catalysts are long molecules, leading to a question of whether they could have been synthesized by a primitive replicator. It is proposed that the efficiency of some small ribozymes may have been augmented by other RNAs acting as transactivators.

Development of a potentially wearable glucose sensor for patients with diabetes mellitus: design and in-vitro evaluation.

A potentially wearable glucose sensor was developed, consisting of an oxygen electrode as detector and a dynamic enzyme perfusion system as selector. The selector is a hollow fibre, which can be placed subcutaneously and dialyses glucose from tissue fluid. In this design the problems of enzyme instability and oxygen limitation might be circumvented. The sensor measures glucose reliably for over two weeks, provided a new 10 ml syringe containing a glucose oxidase solution is connected to the system each day.

Observations on the Tullio phenomenon.

Vestibular responses (vertigo, nystagmus-like eye movements) to acoustic stimuli are known as the "Tullio phenomenon". Detailed electro-oculographic analysis of this reaction, as observed in a 30-year-old patient, revealed the following: a maximum amplitude of eye movement (mainly vertical) was achieved by sine wave bursts of high intensity, a frequency of 500 to 1000 Hz and a duration of 100 ms. The ocular deviation was composed of a fast initial component, followed by a slower resetting movement that was often divided into two parts of different velocities. At longer stimulus durations (more than 100 ms) the electro-oculogram showed a fractionation of the eye deviation, terminating in an "off-response". Various positions of the patient's head influenced the direction of the eye motion. The possibility that the Tullio phenomenon may be due to an abnormal excitation of the statolith organs is discussed.

Calibration of a wearable glucose sensor.

Calibration of glucose sensors proved difficult for electrodes with immobilized glucose-oxidase. The correlation between the sensitivity of the electrodes in vitro and in vivo appeared to be poor. We developed a new type of glucose sensor, based on a microdialysis system, in which an oxygen electrode is used as detector outside the body and the enzyme glucose-oxidase dissolved in water is used as a dynamic selector. The enzyme solution is pumped through a hollow fiber placed subcutaneously, before the fluid passes the detector. The glucose sensor was tested in the subcutaneous abdominal tissue of 12 healthy volunteers and 12 type I diabetic patients. Blood glucose was clamped at two levels to permit a two-point calibration of the sensor in vivo. These values correlated well with the in vitro calibration factors (r = 0.949). In subcutaneous tissue the sensor measures 43 +/- 9% of the blood glucose value, using the in vitro calibration factor. No differences were detected between healthy volunteers and diabetic patients.

Development of a wearable glucose sensor; studies in healthy volunteers and in diabetic patients.

A glucose sensor with a subcutaneous dialysis system was tested in six healthy volunteers during an oral glucose tolerance test and in ten diabetic patients with hyperglycemia during rapid decline of blood glucose levels. There was a good correlation between sensor and blood glucose values. During oral glucose tolerance tests in the volunteers, there was a mean delay of 4.4 minutes in the rise of the value registered subcutaneously and of 8.2 minutes in the fall of the curves. In the diabetic patients the maximum delay was 22 minutes. Nine days after insertion of the dialysis system it was still functioning well.

Electroforming of large mirrors.

Thin metal mirrors are prime candidates for space applications, because of their high thermal conductivity, low weight, and ability to withstand vibration during launch. Of all the fabrication processes, at the present state of the art, electroforming appears the most suitable for producing accurate, thin, metal mirrors with satisfactory mechanical and physical properties. Great strides were made over the past two years in precision electroforming. These are described along with General Electric's spincasting process for the fabrication of masters.

A novel function of Escherichia coli transfer RNA nucleotidyltransferase. Biosynthesis of the C-C-A sequence in a phage T4 transfer RNA precursor.

The biosynthesis of the phage T4-coded proline and serine transfer RNA species proceeds through a precursor RNA containing both tRNA sequences. Neither tRNA sequence in the precursor RNA contains the 3'-terminal C-C-A common to all mature tRNAs. Seidman and McClain ((1975) Proc. Natl. Acad. Sci. U. S. A. 72, 1491-1495) have proposed that the C-C-A sequence is added to serine tRNA while it is still part of the large precursor RNA. In the present work, I show that, in vitro, a purified preparation of Escherichia coli tRNA nucleotidyltransferase (EC 2.7.7.25) accurately synthesized 3'-terminal C-C-A in the serine tRNA portion of the precursor RNA. This result establishes a role of tRNA nucleotidyltransferase in the biosynthesis of the phage T4 serine tRNA. The finding that tRNA nucleotidyltransferase utilizes the large precursor RNA as a substrate represents a novel function of the enzyme.