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Biomedical subjects

V Nowotny

Publications and source records attributed to V Nowotny.

At least 19 recordsLinked to original sources
PubMed2001

Association studies using novel polymorphisms in BACE1 and BACE2.

The release of amyloid-beta peptide (Abeta) from beta-amyloid precursor protein (APP) requires cleavage by beta- and gamma-secretases. Several groups have identified a candidate for the beta-site APP-cleaving enzyme, BACE1, and its homologue BACE2. We sequenced the genes for BACE1 and BACE2 and found several polymorphisms in both genes. Genotyping a large cohort of AD cases and controls has shown no association between AD and the intronic polymorphism in BACE2 while there was a weak association between the BACE1 polymorphism in exon 5 and AD in those carrying the APOE epsilon4 allele.

Aged↗
PubMed2001

Probabilistic approaches to the use of higher order clone relationships in physical map assembly.

UNLABELLED: Physical map assembly is the inference of genome structure from experimental data. Map assembly depends on the integration of diverse data including sequence tagged site (STS) marker content, clone sizing, and restriction digest fingerprints (RDF). As experimentally measured data, these are uncertain and error prone. Physical map assembly from error free data is straightforward and can be accomplished in linear time in the number of clones, but the assembly of an optimal map from error prone data is an NP-hard problem. We present an alternative approach to physical map assembly that is based on a probabilistic view of the data and seeks to identify those features of the map that can be reliably inferred from the available data. With this approach, we achieve a number of goals. These include the use of multiple data sources, appropriate representation of uncertainties in the underlying data, the use of clone length information in fingerprint map assembly, and the use of higher order information in map assembly. By higher order information, we mean relationships that are not expressible in terms of neighbouring clone relationships. These include triplet and multiple clone overlaps, the uniqueness of STS position, and fingerprint marker locations. In a probabilistic view of physical mapping, we assert that all of the many possible map assemblies are equally likely a priori. Given experimental data, we can only state which assemblies are more likely than others given the experimental observations. Parameters of interest are then derived as likelihood weighted averages over map assemblies. Ideally these averages should be sums or integrals over all possible map assemblies, but computationally this is not feasible for real-world map assembly problems. Instead, sampling is used to asymptotically approach the desired parameters. Software implementing our probabilistic approach to mapping has been written. Assembly of mixed RDF and STS maps containing up to 60 clones can be accomplished on a desktop PC with run times under an hour. AVAILABILITY: http://stl.wustl.edu/software/gibbsmap/.

Cloning, Molecular↗
PubMed2000

Posttranslational modification and plasma membrane localization of the Drosophila melanogaster presenilin.

Mutations in two genes, presenilin 1 (PS1) and presenilin 2, are linked to early onset cases of familial Alzheimer's disease. The presenilins are thought to contribute to the pathogenesis of Alzheimer's disease by directly or indirectly affecting the proteolytic processing of the amyloid precursor protein. They have also been implicated in the proteolytic processing of Notch. In PS1-deficient mammalian cells, the proteolytic release of the Notch intracellular domain is reduced. Likewise, loss-of-function mutations in Drosophila presenilin (Psn) prevent the production of the intracellular Notch signaling fragment and lead to phenotypes resembling Notch mutants. Here we characterize the Drosophila Psn protein and demonstrate that it undergoes a proteolytic cleavage. We describe Psn expression at different developmental stages of the fly and show Psn localization near both apical and basal plasma membranes. Furthermore, we demonstrate that portions of the Psn protein span the plasma membrane in S2 cells.

Alternative Splicing↗
PubMed1999

Construction of a detailed physical and transcript map of the FTDP-17 candidate region on chromosome 17q21.

Frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17) is an autosomal dominant condition clinically characterized by behavioral, cognitive, and motor disturbances. Until now, at least 13 different FTDP-17 families that show linkage to chromosome 17q21 have been described. To characterize the FTDP-17 candidate region, flanked by the markers D17S1789 and D17S1804, we constructed a physical map in P1 and PAC clones. A detailed transcript map was generated by positioning known genes and EST clusters to the physical map. In total, we investigated 150 STSs mapped to this region. In addition, novel transcripts were isolated by exon-trapping. We were able to localize 19 known genes and a number of ESTs to this chromosomal region. Furthermore, seven novel genes were identified for which we isolated the full-length sequence.

Base Sequence↗
PubMed1997

X chromosome map at 75-kb STS resolution, revealing extremes of recombination and GC content.

A YAC/STS map of the X chromosome has reached an inter-STS resolution of 75 kb. The map density is sufficient to provide YACs or other large-insert clones that are cross-validated as sequencing substrates across the chromosome. Marker density also permits estimates of regional gene content and a detailed comparison of genetic and physical map distances. Five regions are detected with relatively high G + C, correlated with gene richness; and a 17-Mb region with very low recombination is revealed between the Xq13.3 [XIST] and Xq21.3 XY homology loci.

Base Composition↗
PubMed1994

Direct shape determination of ribosomal proteins in solution and within the ribosome by means of neutron scattering.

Following the 'strategy of the glassy ribosome' single protonated ribosomal proteins (r-proteins) were reconstituted into deuterated 50S subunits of Escherichia coli. The deuteration of both rRNA and r-proteins were individually adjusted to such a degree that the ribosomal matrix appeared nearly homogeneous with respect to coherent neutron scattering and had a scattering density equivalent to a D2O solution of about 90%. Neutron scattering of ribosomal subunits was recorded in reconstitution buffer containing three different concentrations of D2O around 90% D2O (contrast variation). The signal-to-noise ratio achieved allowed us to make a direct determination of the radii of gyration of r-proteins within the 50S subunit and thus provides the first information relating to the shape of these proteins in situ. We present the radii of gyration of 11 r-proteins incorporated into 50S subunits and of 9 isolated r-proteins in solution. In addition, the data concerning the overall dimensions of the r-proteins we report on indicate that conformational changes of at least two individual r-proteins occur during the assembly process of the ribosome.

Escherichia coli↗
PubMed1994

High-throughput rapid yeast DNA extraction. Application to yeast artificial chromosomes as polymerase chain reaction templates.

An automation-assisted method enables DNA extraction of over a thousand yeast colonies in a day by one person. Yeast DNA is extracted essentially quantitatively in three steps: (a) cells are converted into spheroplasts with yeast lytic enzyme, (b) cells are lysed with proteinase K, and (c) DNA is collected by ethanol precipitation. The DNA stored at 4 degrees C is active in polymerase chain reaction experiments for more than a year.

Chromosomes, Artificial, Yeast↗
PubMed1993

Alu and L1 sequence distributions in Xq24-q28 and their comparative utility in YAC contig assembly and verification.

The contents of Alu- and L1-containing TaqI restriction fragments were assessed by Southern blot analyses across YAC contigs already assembled by other means and localized within Xq24-q28. Fingerprinting patterns of YACs in contigs were concordant, and using software based on that of M. V. Olson et al. (1986, Proc. Natl. Acad. Sci. USA 83: 7826) to analyze digitized data on fragment sizes, fingerprinting itself could establish matches among about 40% of a test group of 435 YACs. At 100-kb resolution, both repetitive elements were found throughout the region, with no apparent enrichment of Alu or L1 in DNA of G compared to that found in R bands. However, consistent with a random overall distribution, delimited regions of up to 100 kb contained clusters of repetitive elements. The local concentrations may help to account for the reported differential hybridization of Alu and L1 probes to segments of metaphase chromosomes.

Chromosome Mapping↗
PubMed1993

Screening yeast artificial chromosome libraries with robot-aided automation.

Screening of collections of yeast artificial chromosomes utilizing the polymerase chain reaction (PCR) requires large numbers of reactions in parallel. Four steps were implemented to reduce the labor involved: (a) The number of initial samples for DNA extractions was decreased by compressing libraries up to 12-fold. (b) DNA extraction from yeast clones was robot assisted. (c) A BIOMEK 1000 station was adapted to pipette samples for PCR assays. (d) Sample preparation was integrated with a temperature cycler constructed to carry out up to 576 reactions in six 8 x 12-well trays. The implementation of these steps increases the number of reactions per person per day by an order of magnitude. In tests with X-chromosome-specific probes, the robot-aided screening recovered all of the clones detected by slower manual methods.

Chromosomes, Artificial, Yeast↗
PubMed1993

PCR buffer optimization with uniform temperature regimen to facilitate automation.

To facilitate PCR(1,2) reactions in large numbers with uniform conditions, the annealing temperature was fixed and the stringency of the reactions was adjusted by optimizing the ion conditions of the reaction. The buffer system is based primarily on Tris (T), ammonium (N), and potassium (K) to adapt assay conditions to different primer pairs. The TNK buffers have permitted successful screening of a 60,000-clone yeast artificial chromosome (YAC) library with more than 200 primer pairs.

Automation↗
PubMed1992

Inter-protein distances within the large subunit from Escherichia coli ribosomes.

Selected pairs of protonated ribosomal proteins were reconstituted into deuterated 50S subunits from Escherichia coli ribosomes. The rRNA of the deuterated ribosomal matrix was derived from cells grown in 76% D2O, the deuterated protein moiety from cells grown in 84% D2O. This procedure warrants that the coherent neutron scattering of deuterated proteins and rRNA is nearly the same and equals that of a D2O solution of approximately 90%. The neutron scattering is recorded in a reconstitution buffer containing approximately 90% D2O. The result is a significant improvement of the coherent signal:noise ratio over traditional methods; due to this dilute solutions can be used, thus preventing unfavorable inter-particle effects. From the diffraction pattern the distance between the mass centers of gravity of the two protonated proteins can be deduced. In this way, 50 distances between proteins within the large subunit have been determined which provide a basis for future models of the large ribosomal subunit describing the spatial distribution of the ribosomal proteins. A model containing seven ribosomal proteins is presented.

Deuterium↗
PubMed1988

Assembly of the 30S subunit from Escherichia coli ribosomes occurs via two assembly domains which are initiated by S4 and S7.

A protein which initiates assembly of ribosomes is defined as a protein which binds to the respective rRNA without cooperativity (i.e., without the help of other proteins) during the onset of assembly and is essential for the formation of active ribosomal subunits. The number of proteins binding without cooperativity was determined by monitoring the reconstitution output of active particles at various inputs of 16S rRNA, in the presence of constant amounts of 30S-derived proteins (TP30): This showed that only two of the proteins of the 30S subunit are assembly-initiator proteins. These two proteins are still present on a LiCl core particle comprising 16S rRNA and 12 proteins (including minor proteins). The 12 proteins were isolated, and a series of reconstitution experiments at various levels of rRNA excess demonstrated that S4 and S7 are the initiator proteins. Pulse-chase experiments performed during the early assembly with 14C- and 3H-labeled TP30 and the determination of the 14C/3H ratio of the individual proteins within the assembled particles revealed a bilobal structure of the 30S assembly: A group of six proteins headed by S4 (namely, S4, S20, S16, S15, S6, and S18) resisted the chasing most efficiently (S4 assembly domain). None of the proteins depending on S7 during assembly were found in this group but rather in a second group with intermediate chasing stability [S7 assembly domain; consisting of S7, S9, (S8), S19, and S3]. A number of proteins could be fully chased during the early assembly and therefore represent "late assembly proteins" (S10, S5, S13, S2, S21, S1). These findings fit well with the 30S assembly map.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗
PubMed1986

Assembly analysis of ribosomes from a mutant lacking the assembly-initiator protein L24: lack of L24 induces temperature sensitivity.

Previously, we have shown that the ribosomal protein L24 is one of two assembly-initiator proteins. L24 is essential for early steps of the assembly of the 50S ribosomal subunit but it is not involved in both the late assembly and the ribosomal functions. Surprisingly, an E. coli mutant (TA109-130) exists which lacks L24. This apparent paradox is analyzed and resolved in this paper. The phenotypic is analyzed and resolved in this paper. The phenotypic features of the mutant lacking L24, are a temperature sensitivity (growth severely reduced beyond 34 degrees C), a very low growth rate already at permissive temperatures (at least six-fold slower than wild type) and an underproduction of 50S subunits (molar ratio of 30S to 50S about 1:0.5). The S value of the mutant large subunits is 47S, and they are normally active in poly(Phe) synthesis. The total protein of the mutant large subunits show negligible activity in the total reconstitution assay using the standard two-step procedure. Number analysis of the assembly-initiator proteins revealed that only one initiator protein is effective, as expected. The activity is restored upon addition of wild-type L24. However, when the temperature of the first step is lowered from 44 degrees to 36 degrees C, reconstitution of active particles occurs with a 50% efficiency in the absence of L24. The recovery of activity is accompanied by the appearance of again two initiator proteins, when the mutant TP50 lacking L24 is used in the reconstitution assay at the 'permissive' temperature of 36 degrees C during the first step.(ABSTRACT TRUNCATED AT 250 WORDS)

Escherichia coli↗
PubMed1983

Shape determinations of ribosomal proteins in situ.

Ribosomes are heterogeneous for neutrons because RNA and proteins have different neutron-scattering-length densities. This heterogeneity is an obstacle to the shape determination of single ribosomal components within the ribosome. Therefore, we homogenized (matched) the neutron-scattering-length densities of RNA and proteins. 23S and 5S RNA from the large ribosomal subunit were isolated from cells grown in a medium containing 76% 2H2O. The total protein fraction of the large ribosomal subunit was isolated from cells grown in a medium containing 84% 2H2O. When these constituents were used for total reconstitution of 50S subunits, neutron scattering measurements of the reconstituted particles revealed excellent matching near 100% 2H2O. A three-step reconstitution procedure was developed that allowed the reconstitution of 50S subunits from deuterated RNA, deuterated total (i.e., unfractionated) proteins, and single protonated proteins. The reconstituted particles contain one protonated protein or two in a matched ribosomal matrix and were used for shape determination or distance measurement of mass centers of gravity, respectively. The signal/noise ratio is high enough to allow measurement in solutions containing nearly 100% 2H2O at concentrations of only 300-500 A260 nm units/ml. Our experiments have proved the feasibility of our biochemical strategy. The shape determinations of ribosomal proteins in situ gave radii of gyration for L1, L3, L4, and L23 of 26 +/- 2, 22 +/- 2, 20 +/- 2, and 13 +/- 2 A, respectively.

Escherichia coli↗
PubMed1982

Initiator proteins for the assembly of the 50S subunit from Escherichia coli ribosomes.

An rRNA-binding protein that binds to the rRNA independently of other proteins during the course of ribosomal assembly is termed "assembly initiator protein." In spite of the large number of rRNA-binding proteins (more than 17 out of 32 proteins have been identified in the case of the large ribosomal subunit), only a very small number of proteins should actually initiate ribosomal assembly for theoretical reasons. Here we demonstrate that only two of the L proteins derived from the large subunit (50S) function as assembly initiator proteins. Two different techniques are used to identify these initiator proteins: reconstitution experiments with purified proteins and pulse-chase experiments during in vitro assembly. Both methods independently identify L24 and L3 as initiator proteins for the 50S assembly. The existence of two initiator proteins (not just one) resolves an apparent contradiction--namely, that on the one hand, rRNA is synthesized in excess under unfavorable growth conditions, whereas on the other hand, rRNA-binding proteins should be available for translational control.

Animals↗