A new reagent for preparing radioactively labeled nucleoside derivatives.

The identification of benzimidazole incorporated into RNA of Escherichia coli as benzimidazole nucleoside by means of mass spectrometry is reported. Trimethylsilylation of an enzymatic digest of bacterial RNA allowed the separation of the different nucleosides by gas chromatography. The coupled mass spectrometer was used as a mass specific detector and allowed the sensitive detection of single components of the complex mixture. Thus, benzimidazole ribonucleoside could be detected in hydrolysates of RNA from E. coli fed benzimidazole in the culture broth, although this nucleoside could not be completely separated from uridine by the gas chromatographic systems explored. Quantitation of the benzimidazole nucleoside content revealed that benzimidazole is incorporated into RNA amounting to 16% relative to adenosine.     

A simple electronic apparatus for the analysis of radioactively labeled gel electrophoretograms

Radioactively labeled gel electrophoretograms are generally analyzed either by slicing the gel and dissolving the individual pieces in a fluor for scintillation counting or by exposure to photographic emulsions. In this report, we describe a technique for fast quantitative analysis of such electrophoretograms, based on counting individually β-or γ-particles emerging from the gel surface and locating their position to ±0.6 mm. The apparatus which employs a position-sensitive single-wire gas proportional counter is simple to construct and operate with readily available electronics.     

Effects of concanavalin A on the migration of radioactively labeled lymphoid cells

The effects of the jackbean globulin Concanaalin A (Con A) on the distribution of radioactive ⁵¹Cr-labeled lymph node cells was studied in CBA mice. Lymph node cells treated in vitro with Con A in subagglutinating noncytotoxic doses were unable to “home” to the lymph nodes of syngeneic recipients after intraenous injection. The effect was almost immediate and seemed unrelated to mitogenesis. The inhibitory effect of Con A on lymphocyte migration could be partially reersed by alpha-methyl mannoside; the degree of migratory impairment was related to the amount of Con A bound to the lymphocyte surface at the time of transfer. The membrane site at which Con A binds to the lymphocytes is similar to that which is bound by heterologous antilymphocyte serum but is probably distinct from the theta antigenic site. These data lend support to the hypothesis that surface lymphocyte carbohydrate determinants are involved in the specific lymphocyte “homing” receptor.     

A rapid, guantitative, and selective estimation of radioactively labeled peptidoglycan in gram-positive bacteria

The Park and Hancock procedure for the isolation of bacterial cell wall peptidoglycans has been modified. The method developed permits rapid and accurate determination of small quantities of radioactively labeled material. The principal modifications consist of carrying out the procedure on glass-fiber filters after a vigorous hydrolysis of the cells with TCA (96°C, 30 min) and using Pronase in place of trypsin.     

Preparation and binding of radioactively labeled porcine transforming growth factor type beta

This report describes the labeling of porcine transforming growth factor type beta (TGF-beta) with 125-iodine. Its binding to NRK cells and three other cell lines has been examined. The data indicate that NRK cells exhibit approximately 10,000 receptors for porcine TGF-beta per cell, with an apparent dissociation constant of 45 pM. The binding of porcine 125I-TGF-beta can be blocked by porcine, murine and human TGF-beta but not by several well characterized growth factors. In all respects examined, the binding observed with porcine 125I-TGF-beta appears to be the same as that observed with human TGF-beta. The findings reported here argue that porcine 125I-TGF-beta can be used to quantitate TGF-beta receptors on a wide range of mammalian cells.     

TomoAlign: A novel approach to correcting sample motion and 3D CTF in CryoET

TomoAlign is a software package that integrates tools to mitigate two important resolution limiting factors in cryoET, namely the beam-induced sample motion and the contrast transfer function (CTF) of the microscope. The package is especially focused on cryoET of thick specimens where fiducial markers are required for accurate tilt-series alignment and sample motion estimation. TomoAlign models the beam-induced sample motion undergone during the tilt-series acquisition. The motion models are used to produce motion-corrected subtilt-series centered on the particles of interest. In addition, the defocus of each particle at each tilt image is determined and can be corrected, resulting in motion-corrected and CTF-corrected subtilt-series from which the subtomograms can be computed. Alternatively, the CTF information can be passed on so that CTF correction can be carried out entirely within external packages like Relion. TomoAlign serves as a versatile tool that can streamline the cryoET workflow from initial alignment of tilt-series to final subtomogram averaging during in situ structure determination.     

A unified association analysis approach for family and unrelated samples correcting for stratification

There are two common designs for association mapping of complex diseases: case-control and family-based designs. A case-control sample is more powerful to detect genetic effects than a family-based sample that contains the same numbers of affected and unaffected persons, although additional markers may be required to control for spurious association. When family and unrelated samples are available, statistical analyses are often performed in the family and unrelated samples separately, conditioning on parental information for the former, thus resulting in reduced power. In this report, we propose a unified approach that can incorporate both family and case-control samples and, provided the additional markers are available, at the same time corrects for population stratification. We apply the principal components of a marker matrix to adjust for the effect of population stratification. This unified approach makes it unnecessary to perform a conditional analysis of the family data and is more powerful than the separate analyses of unrelated and family samples, or a meta-analysis performed by combining the results of the usual separate analyses. This property is demonstrated in both a variety of simulation models and empirical data. The proposed approach can be equally applied to the analysis of both qualitative and quantitative traits.     

A novel approach to enhance antibody sensitivity and specificity by peptide cross-linking

Most current techniques employed to improve antigen-antibody signals in Western blotting and in immunohistochemistry rely on sample processing prior to staining (e.g., microwaving) or using a more robust reporter (e.g., a secondary antibody with biotin-streptavidin). We have developed and optimized a new approach intended to stabilize the complexes formed between antigens and their respective primary antibodies by cupric ions at high pH. This technique improves the affinity and lowers cross-reactivity with nonspecific bands of approximately 20% of antibodies tested (5/25). Here we report that this method can enhance antigen-antibody specificity and can improve the utility of some poorly reactive primary antibodies.     

Rapid routes of synthesis of chemically reactive and highly radioactively labeled alpha- and beta-oligonucleotide derivatives for in vivo studies

Development of the antisense oligonucleotide strategy for the regulation of gene expression in vivo poses several problems: the stability of oligonucleotides toward intracellular nucleases, labeling of oligonucleotides with high specific radioactivity, improvements of penetration of oligonucleotides into living cells, and enhancement of antisense action by coupling of chemically active groups. In the present paper synthesis of highly radioactively labeled [32P]- and [35S]oligonucleotide derivatives is described starting from both natural (beta) and nuclease-resistant (alpha) anomers of oligonucleotides. Conditions for preparative phosphorylation and thiophosphorylation suitable for oligonucleotides of various lengths, base composition, and anomeric forms were established. The stability of the phosphoramide bond under in vivo experimental conditions was checked. The methods of terminal phosphate chemical activation and terminal thiophosphate alkylation were applied to synthesize oligonucleotides equipped with hydrophobic, intercalating, alkylating, and photoactivatable groups. In the case of porphyrin-oligonucleotide conjugates, a series of new monofunctional porphyrin derivatives bearing a free aliphatic amino group was developed.     

A pseudolikelihood approach to correcting for ascertainment bias in family studies.

The Cannings and Thompson approach to correcting for ascertainment bias in family studies is extended to settings with multiple ascertainment. The extension is based on maximizing a pseudolikelihood. Two approaches to computing standard errors for the maximum pseudolikelihood estimate are described. One is especially simple to compute, while the other is more generally applicable. Simulation experiments suggest that the standard-error computations can be quite accurate.     

Hybribox: a device for processing numbers of radioactively labeled hybridization filters with a minimum of personal exposure

The device described permits the rapid and efficient processing of large numbers of filter hybridizations while virtually eliminating exposure to radioactive emission from the labeled probe. The filters are annealed and washed without being transferred from the holder. Throughout the process, the Plexiglas composition of the device absorbs the beta particles and exposure to the hands and arms is essentially zero. Since the hybridization solution can be reused several times, the cost involved approximates that of the conventional methods for hybridization.     

Binding of radioactively labeled carboxyatractyloside, atractyloside and bongkrekic acid to the ADP translocator of potato mitochondria

1. 1. The inhibition of the ADP-stimulated respiration of potato mitochondria by carboxyatractyloside is relieved by high concentration of ADP or by the uncoupler carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP). Atractyloside is a much less potent inhibitor than carboxyatractyloside. The inhibition of the ADP-stimulated respiration required about 60-times more atractyloside than carboxyatractyloside.

2. 2. [³⁵S]carboxyatractyloside and [³H]bongkrekic acid bind to potato mitochondria with high affinity (K_d = 10 to 20 nM, n = 0.6–0.7 nmol per mg protein). Added ADP competes with carboxyatractyloside for binding; on the contrary ADP increases the amount of bound bongkrekic acid. [³H]atractyloside binds to potato mitochondria with a much lower affinity (K_d = 0.45 μM) than carboxyatractyloside or bongkrekic acid.

3. 3. Bound [³H]atractyloside is displaced by ADP, carboxyatractyloside and bongkrekic acid. The displacement of bound [³⁵S]carboxyatractyloside by bongkrekic acid and of bound [³H]bongkrekic acid by carboxyatractyloside is markedly increased by ADP.

4. 4. Bongkrekic acid competes with [³⁵S]carboxyatractyloside for binding. Addition of a small concentration of ADP considerably enhances the inhibitory effect of bongkrekic acid on [³⁵S]carboxyactratyloside binding.

5. 5. The adenine nucleotide content of potato mitochondria is of the order of 1 nmol per mg protein. ADP transport in potato mitochondria is inhibited by atractyloside 30- to 40-times less efficiently than by carboxyatractyloside.

A hill-climbing approach for automatic gridding of cDNA microarray images

Image and statistical analysis are two important stages of cDNA microarrays. Of these, gridding is necessary to accurately identify the location of each spot while extracting spot intensities from the microarray images and automating this procedure permits high-throughput analysis. Due to the deficiencies of the equipment used to print the arrays, rotations, misalignments, high contamination with noise and artifacts, and the enormous amount of data generated, solving the gridding problem by means of an automatic system is not trivial. Existing techniques to solve the automatic grid segmentation problem cover only limited aspects of this challenging problem and require the user to specify the size of the spots, the number of rows and columns in the grid, and boundary conditions. In this paper, a hill-climbing automatic gridding and spot quantification technique is proposed which takes a microarray image (or a subgrid) as input and makes no assumptions about the size of the spots, rows, and columns in the grid. The proposed method is based on a hill-climbing approach that utilizes different objective functions. The method has been found to effectively detect the grids on microarray images drawn from databases from GEO and the Stanford genomic laboratories.     

Basic properties and information theory of Audic-Claverie statistic for analyzing cDNA arrays

Background  

The Audic-Claverie method [1] has been and still continues to be a popular approach for detection of differentially expressed genes in the SAGE framework. The method is based on the assumption that under the null hypothesis tag counts of the same gene in two libraries come from the same but unknown Poisson distribution. The problem is that each SAGE library represents only a single measurement. We ask: Given that the tag count samples from SAGE libraries are extremely limited, how useful actually is the Audic-Claverie methodology? We rigorously analyze the A-C statistic that forms a backbone of the methodology and represents our knowledge of the underlying tag generating process based on one observation.     

A unified approach for quantifying, testing and correcting population stratification in case-control association studies

The HapMap project has given case-control association studies a unique opportunity to uncover the genetic basis of complex diseases. However, persistent issues in such studies remain the proper quantification of, testing for, and correction for population stratification (PS). In this paper, we present the first unified paradigm that addresses all three fundamental issues within one statistical framework. Our unified approach makes use of an omnibus quantity (delta), which can be estimated in a case-control study from suitable null loci. We show how this estimated value can be used to quantify PS, to statistically test for PS, and to correct for PS, all in the context of case-control studies. Moreover, we provide guidelines for interpreting values of delta in association studies (e.g., at alpha = 0.05, a delta of size 0.416 is small, a delta of size 0.653 is medium, and a delta of size 1.115 is large). A novel feature of our testing procedure is its ability to test for either strictly any PS or only 'practically important' PS. We also performed simulations to compare our correction procedure with Genomic Control (GC). Our results show that, unlike GC, it maintains good Type I error rates and power across all levels of PS.     

A novel method for increasing the expression level of recombinant proteins

Expression of recombinant proteins is an important step towards elucidating the functions of many genes discovered through genomic sequencing projects. It is also critical for validating gene targets and for developing effective therapies for many diseases. Here we describe a novel method to express recombinant proteins that are extremely difficult to produce otherwise. The increased protein expression level is achieved by using a fusion partner, MTB32-C, which is the carboxyl terminal fragment of the Mycobacterium tuberculosis antigen, MTB32 (Rv0125). By fusing MTB32-C to the N-termini of target genes, we have demonstrated significant enhancement of recombinant protein expression level in Escherichia coli. The inclusion of a 6xHis tag and the 128-amino acid of MTB32-C will add 13.5 kDa to the fusion molecule. Comparison of the mRNA levels of the fusion and non-fusion proteins indicated that the increased fusion protein expression may be regulated at translational or post-translational steps. There are many potential applications for the generated fusion proteins. For example, MTB32-C fusion proteins have been used successfully as immunogens to generate both polyclonal and monoclonal antibodies. These antibodies have been used to characterize cellular localization of the proteins and to validate gene targets at protein level. In addition, these antibodies may be useful in diagnostic and therapeutic applications for many diseases. If desired, the MTB32-C portion in the fusion protein can be removed after protein expression, making it possible to study protein structure and function as well as to screen for potential drugs. Thus, this novel fusion expression system has become a powerful tool for many applications.