Biomedical subjects

Z Modrusan

Publications and source records attributed to Z Modrusan.

7 recordsLinked to original sources

PubMed2001

The Arabidopsis BELL1 and KNOX TALE homeodomain proteins interact through a domain conserved between plants and animals.

Interactions between TALE (three-amino acid loop extension) homeodomain proteins play important roles in the development of both fungi and animals. Although in plants, two different subclasses of TALE proteins include important developmental regulators, the existence of interactions between plant TALE proteins has remained unexplored. We have used the yeast two-hybrid system to demonstrate that the Arabidopsis BELL1 (BEL1) homeodomain protein can selectively heterodimerize with specific KNAT homeodomain proteins. Interaction is mediated by BEL1 sequences N terminal to the homeodomain and KNAT sequences including the MEINOX domain. These findings validate the hypothesis that the MEINOX domain has been conserved between plants and animals as an interaction domain for developmental regulators. In yeast, BEL1 and KNAT proteins can activate transcription only as a heterodimeric complex, suggesting a role for such complexes in planta. Finally, overlapping patterns of BEL1 and SHOOT MERISTEMLESS (STM) expression within the inflorescence meristem suggest a role for the BEL1-STM complex in maintaining the indeterminacy of the inflorescence meristem.

Arabidopsis↗

PubMed2001

Optimized T7 amplification system for microarray analysis.

Glass cDNA microarray technologies offer a highly parallel approach for profiling expressed gene sequences in disease-relevant tissues. However, standard hybridization and detection protocols are insufficient for milligram quantities of tissue, such as those derived from needle biopsies. Amplification systems utilizing T7 RNA polymerase can provide multiple cRNA copies from mRNA transcripts, permitting microarray studies with reduced sample inputs. Here, we describe an optimized T7-based amplification system for microarray analysis that yields between 200- and 700-fold amplification. This system was evaluated with both mRNA and total RNA samples and provided microarray sensitivity and precision that are comparable to our standard production process without amplification. The size distributions of amplified cRNA ranged from 200 bp to 4 kb and were similar to original mRNA profiles. These amplified cRNA samples were fluorescently labeled by reverse transcription and hybridized to microarrays comprising approximately 10,000 cDNA targets using a dual-channel format. Replicate hybridization experiments were conducted with the same and different tissues in each channel to assess the sensitivity and precision of differential expression ratios. Statistical analysis of differential expression ratios showed the lower limit of detection to be about 2-fold within and between amplified data sets, and about 3-fold when comparing amplified data to unamplified data (99.5% confidence).

Biotechnology↗

PubMed2000

CPT-EIA assays for the detection of vancomycin resistant vanA and vanB genes in enterococci.

Cycling Probe Technology (CPT) was combined with a colorimetric enzyme-immuno assay (EIA) to develop two assays for the detection of vanA and vanB genes in vancomycin resistant enterococci (VRE). The CPT-EIA assay employs a gene-specific fluorescein labeled DNA-RNA-DNA probe that gets cleaved within the probe : target duplex. The cleaved DNA probe fragments dissociate from the target, making it available for further cycling. Following the separation of cleaved probe fragments, anti-fluorescein-horseradish peroxidase antibodies are used for the detection of uncleaved probes. The two CPT-EIA assays were used to screen a collection of 440 clinical isolates (Modrusan et al., 1999). All of the 154 VanA and 131 VanB isolates were correctly identified in the vanA and vanB CPT-EIA, respectively. The VanA and VanB isolates were differentiated from vancomycin sensitive enterococci (VSE) and also from the VanC isolates. In addition, an accurate VRE detection in the CPT-EIA assay was shown with cultures grown on eight different media.

Anti-Bacterial Agents↗

PubMed2000

Rapid solid-phase immunoassay for detection of methicillin-resistant Staphylococcus aureus using cycling probe technology.

A Cycling Probe Technology (CPT) assay with a lateral-flow device (strip) was developed for the detection of the mecA gene from methicillin-resistant Staphylococcus aureus (MRSA) cultures. The assay uses a mecA probe (DNA-RNA-DNA) labeled with fluorescein at the 5' terminus and biotin at the 3' terminus. The CPT reaction occurs at a constant temperature, which allows the probe to anneal to the target DNA. RNase H cuts the RNA portion of the probe, allowing the cleaved fragments to dissociate from the target DNA, making the target available for further cycling. The strip detection step uses a nitrocellulose membrane with streptavidin and immunoglobulin G antibody impregnated on the surface. In the absence of the mecA gene, the uncut probe is bound to an antifluorescein-gold conjugate and is then captured by the streptavidin to form a test line. In the presence of the mecA gene, the probe is cut and no test line is formed on the strip. A screen of 324 S. aureus clinical isolates by the CPT-strip assay showed a 99.4% sensitivity and a 100% specificity compared to the results of PCR for the detection of the mecA gene. Specificity testing showed that the CPT-strip assay did not exhibit any cross-reactivity with a panel of mecA-negative non-S. aureus isolates. The CPT-strip assay is simple and does not require sophisticated equipment. Furthermore, the assay takes 1.5 h starting from a primary culture to the time to detection of the mecA gene in S. aureus isolates.

Bacterial Proteins↗

PubMed1999

Detection of vancomycin resistant genes vanA and vanB by cycling probe technology.

Cycling Probe Technology (CPT) has been used to develop gene-based assays for detection of vancomycin resistance genes vanA and vanB in enterococci (VRE). Cycling Probe Technology utilizes a chimeric DNA-RNA-DNA probe that is cleaved by the enzyme RNase H when hybridized to its complementary DNA target. Conversion of full-length probe into the cleaved probe fragments is the basis for detection and quantification of the CPT reaction. Two gene-specific probes, each one unique to either the vanA or vanB gene, were utilized for development of vanA and vanB CPT assays, respectively. Both vanA and vanB CPT assays were used to determine the presence or absence of the corresponding gene in 440 clinical enterococcal isolates. The presence of vanA and vanB gene sequences was detected in 154 and 131 isolates, respectively. Phenotypic characterization of all isolates was determined through interpretation of conventional susceptibility data obtained with the disk diffusion method. Comparison between disk diffusion characterization and CPT assays revealed 11 discrepant isolates. The identity of these isolates was resolved by polymerase chain reaction (PCR) which confirmed the vanA and vanB CPT assay data. Therefore, compared to conventional phenotyping, both the vanA and vanB CPT assays appeared superior for accurate identification of VanA and VanB isolates.

Bacterial Proteins↗

PubMed1998

Spermine-mediated improvement of cycling probe reaction.

Cycling probe technology (CPT) represents a simple method for detection of DNA target sequences. Cycling probe technology utilizes a chimeric DNA-RNA-DNA probe which is cleaved by RNase H when hybridized with its complementary target. Probe cleavage in the presence or absence of target generates CPT product or background, respectively. Addition of non-homologous DNA into the CPT reaction affects the background and CPT product. Low amounts of human DNA (4-40 ng) result in high background while higher amounts (40-400 ng) inhibit the reaction. The simultaneous addition of spermine and EGTA into the CPT reaction containing human DNA resulted in a significant release of the inhibition and a reduction of background. The presence of spermine alone caused an increase of probe cleavage whereas addition of EGTA increased the specificity of the CPT. A possible mechanism by which spermine could lead to this improvement of CPT has been proposed. Using a membrane-binding assay, the authors demonstrated that human DNA competes with the probe for binding to RNase H. Furthermore, by using a DNA-agarose column, it has been shown that such RNase H-DNA binding can be disrupted by spermine. Within the CPT reaction, similar spermine-mediated displacement of RNase H from human DNA could lead to an improved CPT efficiency.

Aurora Kinases↗

PubMed1995

The BELL1 gene encodes a homeodomain protein involved in pattern formation in the Arabidopsis ovule primordium.

Ovule development in Arabidopsis involves the formation of three morphologically defined proximal-distal pattern elements. Integuments arise from the central pattern element. Analysis of Bell 1 (Bel 1) mutant ovules indicated that BEL1 was required for integument development. Cloning of the BEL1 locus reveals that it encodes a homeodomain transcription factor. Prior to integument initiation, BEL1 RNA localizes to the central domain, providing molecular evidence for a central pattern element. Therefore, proximal-distal patterning of the ovule involves the regulated expression of the BEL1 gene that controls integument morphogenesis. A model for BEL1 function is evaluated with regard to new data showing the expression pattern of the floral homeotic gene AGAMOUS (AG) early in wild-type and BEL1 ovule development.

AGAMOUS Protein, Arabidopsis↗