Preliminary investigations of ultrasound induced acoustic streaming using particle image velocimetry.

Particle image velocimetry was used to investigate ultrasound-induced acoustic streaming in a system for the enhanced uptake of substances from the aquatic medium into fish. Four distinct regions of the induced streaming in the system were observed and measured. One of the regions was identified as an preferential site for substance uptake, where the highest velocities in proximity to the fish surface were measured. A positive linear relationship was found between the ultrasound intensity and the maximum streaming velocity, where a unitless geometric factor, specific to the system, was calculated for correcting the numerical relationship between the two parameters. The results are part of a comprehensive study aimed at improving mass transdermal administrations of substances (e.g. vaccines, hormones) into fish from the aquatic medium.

DENS (differential extension with nucleotide subsets): application to the sequencing of human genomic DNA and cDNA.

Here we describe further development of our method of DNA sequencing by Differential Extension with Nucleotide Subsets (DENS) and its application to the sequencing of human genomic DNA and full-insert cDNA. Essentially, DENS is primer walking without custom primer synthesis; instead, DENS uses a presynthesized library of octamer primers degenerate in two positions (4,096 tubes/sequences for a complete library). DENS converts an octamer selected from this library into a long primer on the template, at the intended site only. This is done using a two-step procedure which starts with a limited extension of the octamer (at 20 degrees C) in the presence of only two of the four possible dNTPs. The primer is extended by five bases or more at the intended priming site, which is deliberately selected to maximize the extension length (as are the two-dNTP set and the primer itself). The subsequent termination reaction at 60 degrees C then accepts the primer extended at the intended site, but not at alternative sites, where the initial extension (if any) is generally much shorter. This paper presents a set of rules for selection of DENS priming sites. We also compare different ways of template preparation for DENS sequencing. The data were obtained from primer walking on three human genomic DNA subclones of 3 to 4 kbp and four cDNA clones containing inserts of 1.9, 2.3, 3.8, and 4.9 kbp. Full-length sequences were obtained from both strands of each subclone by automated dye-terminator fluorescent DNA sequencing using DENS with degenerate octamer primers. We compared the following types of DNA templates: single-stranded and double-stranded phagemid DNA, double-stranded PCR products, asymmetric PCR products, and single-stranded DNA produced by digestion with Lambda Exonuclease of double-stranded PCR product phosphorylated at one end (Exo-PCR). While all of the preps were found to work, the best results were obtained with Exo-PCR and phagemid single-stranded DNA. Exo-PCR directly from overnight bacterial culture with no plasmid prep of any kind yielded templates for DENS as good as Exo-PCR from purified DNA. We found that the Tm of the differentially extended octamers is an important factor in the success of DENS. Clustering of successful reactions was clearly distinguished in the Tm range of 50-66 degrees C, with success rates of 70% for Exo-PCR and 65% for ss phagemid templates.

Interdependence between DNA template secondary structure and priming efficiencies of short primers.

Here we analyze the effect of DNA folding on the performance of short primers and describe a simple technique for assessing hitherto uncertain values of thermodynamic parameters that determine the folding of single-stranded DNA into secondary structure. An 8mer with two degenerate positions is extended simultaneously at several complementary sites on a known template (M13mp18) using one, two or three (but never all four) of the possible dNTPs. The length of the extension is site specific because it is limited by the first occurrence in the downstream template sequence of a base whose complementary dNTP is not present. The relative priming efficiencies of different sites are then ranked by comparing their band brightnesses on a gel. The priming efficiency of a short primer (unlike conventional long primers) depends dramatically on the secondary structure of the template at and around the priming site. We calculated the secondary structure and its effect on priming using a simple model with relatively few parameters which were then optimized to achieve the best match between the predictions and the actual rankings of the sites in terms of priming efficiency. This work introduces an efficient and conceptually novel approach that in the future can make use of more data to optimize a larger set of DNA folding parameters in a more refined model. The model we used, however crude it may be, significantly improved the prediction of priming efficiencies of 8mer primers and appreciably raised the success rate of our DNA sequencing technique (from 67 to 91% with a significance of P < 7 x 10(-5)), which uses such primers.

Association of yeast SIN1 with the tetratrico peptide repeats of CDC23.

The yeast SIN1 protein is a nuclear protein that together with other proteins behaves as a transcriptional repressor of a family of genes. In addition, sin1 mutants are defective in proper mitotic chromosome segregation. In an effort to understand the basis for these phenotypes, we employed the yeast two-hybrid system to identify proteins that interact with SIN1 in vivo. Here we demonstrate that CDC23, a protein known to be involved in sister chromatid separation during mitosis, is able to directly interact with SIN1. Furthermore, using recombinant molecules in vitro, we show that the N terminal of SIN1 is sufficient to bind a portion of CDC23 consisting solely of tetratrico peptide repeats. Earlier experiments identified the C-terminal domain of SIN1 to be responsible for interaction with a protein that binds the regulatory region of HO, a gene whose transcription is repressed by SIN1. Taken together with the results presented here, we suggest that SIN1 is a chromatin protein having at least a dual function: The N terminal of SIN1 interacts with the tetratrico peptide repeat domains of CDC23, a protein involved in chromosome segregation, whereas the C terminal of SIN1 binds proteins involved in transcriptional regulation.

Association of yeast SAP1, a novel member of the 'AAA' ATPase family of proteins, with the chromatin protein SIN1.

The yeast SIN1 protein is a nuclear protein that together with other proteins behaves as a transcriptional repressor of a family of genes. In addition, sin1 mutants are defective in proper mitotic chromosome segregation. In an effort to understand the basis for these phenotypes, we employed the yeast two-hybrid system to identify proteins that interact with SIN1 in vivo. Here, we demonstrate that SAP1, a novel protein belonging to the 'AAA' family of ATPases, is able to directly interact with SIN1. Furthermore, we show, using recombinant molecules in vitro, that a short 27 amino acid sequence near the N-terminal of SIN1 is sufficient to bind SAP1. Previous experiments defined different domains of SIN that interact with other proteins and with DNA. The C-terminal domain of SIN1 was shown to be responsible for interaction with a protein that binds the regulatory region of HO, a gene whose transcription is repressed by SIN1. The central 'HMG1-like region' of SIN1 binds DNA, while the N-terminal of SIN1 can bind CDC23, a protein that regulates chromosome segregation. These data, taken together with the results presented here, suggest that SIN1 is a multifunctional chromatin protein that can interact with a number of different proteins that are involved in several different cellular functions.

[A microprocessor monitoring system for control of the status of the fetus]. / Mikroprotsessornaia monitornaia sistema kontrolia sostoianiia ploda.

To estimate the fetus condition according to its cardiotocogram it is very important to improve the accuracy of measurements of its parameters and objectifications of the estimate criteria. The described microprocessor system helps to meet these requirements. The described microprocessor system allows to measure immediate fetus contractions with the help of the autocorrelation method and automatic measurements of the cardiotocogram parameters.