Biomedical subjects

J H Hegemann

Publications and source records attributed to J H Hegemann.

At least 19 recordsLinked to original sources

PubMed2002

The vesicular transport protein Cgp1p/Vps54p/Tcs3p/Luv1p is required for the integrity of the actin cytoskeleton.

The CGP1 gene was identified in a screen for mutations that were synthetic lethal in combination with a deletion of the gene (CPF1) for centromere and promoter factor 1. Cells deleted for CGP1 showed reduced viability, were temperature sensitive for growth and exhibited altered sensitivity to microtubule-destabilizing drugs. Furthermore, Deltacgp1 cells showed increased rates of loss of a circular minichromosome and defects in the positioning of the short mitotic spindle. Further phenotypic analysis of Deltacgp1 cells revealed that loss of Cgp1p function led to severe depolarization of the actin cytoskeleton. In addition, cells deleted for CGP1 were hypersensitive to the actin-disrupting compound Latrunculin-A, exhibited strongly reduced polarized localization of the unconventional myosin Myo2p, and showed defects in other actin-related processes, such as shmoo formation and cell wall integrity. Cgp1p was recently identified by several groups as Vps54p, which is a member of the VFT complex that is involved in vesicular protein transport at the level of the late Golgi, acting as a tethering factor. Our data show for the first time that Cgp1p/Vps54p links aspects of vesicular protein transport with the organization of the actin cytoskeleton.

Actins↗

PubMed2002

A second set of loxP marker cassettes for Cre-mediated multiple gene knockouts in budding yeast.

Heterologous markers are important tools required for the molecular dissection of gene function in many organisms, including Saccharomyces cerevisiae. Moreover, the presence of gene families and isoenzymes often makes it necessary to delete more than one gene. We recently introduced a new and efficient gene disruption cassette for repeated use in budding yeast, which combines the heterologous dominant kan(r) resistance marker with a Cre/loxP-mediated marker removal procedure. Here we describe an additional set of four completely heterologous loxP-flanked marker cassettes carrying the genes URA3 and LEU2 from Kluyveromyces lactis, his5(+) from Schizosaccharomyces pombe and the dominant resistance marker ble(r) from the bacterial transposon Tn5, which confers resistance to the antibiotic phleomycin. All five loxP--marker gene--loxP gene disruption cassettes can be generated using the same pair of oligonucleotides and all can be used for gene disruption with high efficiency. For marker rescue we have created three additional Cre expression vectors carrying HIS3, TRP1 or ble(r) as the yeast selection marker. The set of disruption cassettes and Cre expression plasmids described here represents a significant further development of the marker rescue system, which is ideally suited to functional analysis of the yeast genome.

3-Isopropylmalate Dehydrogenase↗

PubMed2001

Heparan sulfate-like glycosaminoglycan is a cellular receptor for Chlamydia pneumoniae.

Chlamydia pneumoniae is an important human intracellular pathogen; however, the pathogenesis of C. pneumoniae infection is poorly understood, and the bacterial adherence mechanism to host cells is unknown. This study examined the role of glycosaminoglycans (GAGs) in the adhesion of C. pneumoniae to eukaryotic cells. Heparin and heparan sulfate were found to inhibit the attachment of C. pneumoniae to human epithelial cells. Reduction in infectivity resulted from the binding of heparin to the organism. Enzymatic removal of heparan sulfate moieties from the host cell surface led to a marked decrease in C. pneumoniae infectivity. Mutant CHO cell lines that were defective in heparan sulfate biosynthesis were less susceptible to C. pneumoniae infection than was the wild-type cell line. However, preincubation of the GAG-deficient CHO cells with exogenous heparin greatly increased infectivity.

Animals↗

PubMed2000

Infection and activation of airway epithelial cells by Chlamydia pneumoniae.

The activation of primary human airway epithelial cells (HAECs) and of the bronchial epithelial cell line BEAS-2B by Chlamydia pneumoniae, an important respiratory pathogen, was characterized. A time-dependent enhanced release of interleukin (IL)-8 and prostaglandin-E(2) and an increased expression of the epithelial adhesion molecule intercellular adhesion molecule-1 (ICAM-1), followed by subsequent transepithelial migration of polymorphonuclear neutrophils (PMN), were also demonstrated. The transepithelial PMN migration could be blocked by an anti-ICAM-1 monoclonal antibody (MAb) but not by MAbs against IL-8. In addition, there was an enhanced C. pneumoniae-mediated activation of NF-kappaB within 30-60 min in HAECs and BEAS-2B, which was followed by increases in mRNA synthesis of IL-8, ICAM-1, and cyclooxygenase-2, with maximal effects occurring 2 h after infection. Thus, C. pneumoniae infects and activates HAECs and BEAS-2B and therefore may be able to trigger a cascade of pro- and anti-inflammatory reactions during chlamydial infections.

Antibodies, Monoclonal↗

PubMed2000

Antibody response to the 60-kDa heat-shock protein of Chlamydia pneumoniae in patients with coronary artery disease.

Serum specimens from 752 individuals undergoing coronary arteriography were examined for antibodies to Chlamydia pneumoniae. Patients with coronary artery disease (CAD) were more likely to have IgG antibodies to C. pneumoniae than were individuals without CAD (60% vs. 52%; P=.007; odds ratio, 1.8; 95% confidence interval, 1. 17-2.77). Antibodies to recombinant hsp60 of C. pneumoniae were found with nearly the same frequency in patients with CAD and individuals without CAD (29% vs. 30%; P=.751). There was no association between chlamydial hsp60 antibodies and the severity of CAD or a previous myocardial infarction. Patient sera reacted most frequently to C. pneumoniae proteins of 17, 38, 40, 58, and 60/62 kDa. Reactivity to these proteins was not different between patients with and without CAD. Study results indicate that neither antibodies to chlamydial hsp60 nor antibodies to other C. pneumoniae proteins are useful for discriminating between seropositive patients with and without CAD.

Antibodies, Bacterial↗

PubMed2000

Molecular analysis of the Saccharomyces cerevisiae YHR076w gene.

Our results demonstrate that open reading frame (ORF) YHR076w on chromosome VIII of Saccharomyces cerevisiae that was previously described as a hypothetical gene is expressed. This ORF is transcribed as an mRNA of approximately 1,100 nucleotides. A 41.2-kDa polypeptide and three others predicted to be modified forms of Yhr076wp are detected by Western blot with a Yhr076wp-specific antibody. Promoter activity assays indicate that YHR076w transcription is regulated by carbon source and primarily by ethanol. Consistent with this observation, we have identified two potential ADR1 regulatory elements in the YHR076w upstream DNA region. Potential YAP1 and HSP elements are also identified in this region, suggesting other forms of regulation. YHR076w knockout strains do not exhibit any measurable growth or morphological phenotype under any conditions tested. However, increased YHR076w gene dosage confers a growth advantage to both wild-type and YHR076w knockout strains on 2% ethanol or 2% galactose medium in a low O2 growth environment. The fluorescence emitted by a Yhr076wp protein fusion to A. aquorin GFP colocalizes with the mitochondria in vivo.

Calmodulin-Binding Proteins↗

PubMed1999

Functional characterization of the S. cerevisiae genome by gene deletion and parallel analysis.

The functions of many open reading frames (ORFs) identified in genome-sequencing projects are unknown. New, whole-genome approaches are required to systematically determine their function. A total of 6925 Saccharomyces cerevisiae strains were constructed, by a high-throughput strategy, each with a precise deletion of one of 2026 ORFs (more than one-third of the ORFs in the genome). Of the deleted ORFs, 17 percent were essential for viability in rich medium. The phenotypes of more than 500 deletion strains were assayed in parallel. Of the deletion strains, 40 percent showed quantitative growth defects in either rich or minimal medium.

Culture Media↗

PubMed1999

Signal transduction pathways activated in endothelial cells following infection with Chlamydia pneumoniae.

Chlamydia pneumoniae is an important respiratory pathogen. Recently, its presence has been demonstrated in atherosclerotic lesions. In this study, we characterized C. pneumoniae-mediated activation of endothelial cells and demonstrated an enhanced expression of endothelial adhesion molecules followed by subsequent rolling, adhesion, and transmigration of leukocytes (monocytes, granulocytes). These effects were blocked by mAbs against endothelial and/or leukocyte adhesion molecules (beta1 and beta2 integrins). Additionally, activation of different signal transduction pathways in C. pneumoniae-infected endothelial cells was shown: protein tyrosine phosphorylation, up-regulation of phosphorylated p42/p44 mitogen-activated protein kinase, and NF-kappaB activation/translocation occurred within 10-15 min. Increased mRNA and surface expression of E-selectin, ICAM-1, and VCAM-1 were noted within hours. Thus, C. pneumoniae triggers a cascade of events that could lead to endothelial activation, inflammation, and thrombosis, which in turn may result in or may promote atherosclerosis.

Blotting, Northern↗

PubMed1999

All 16 centromere DNAs from Saccharomyces cerevisiae show DNA curvature.

All 16 centromere DNA regions of Saccharomyces cerevisiae including 90 bp framing sequences on either side were cloned. These 300 bp long centromere regions were analysed by native polyacrylamide gel electrophoresis and found to display a reduced mobility indicative of DNA curvature. The degree of curvature is centromere dependent. The experimental data were confirmed by computer analysis of the 3-dimensional structure of the CEN DNAs. Altogether these data provide further evidence for a model for budding yeast centromeres in which CEN DNA structure could be important for the assembly, activity and/or regulation of the centromere protein-DNA complex.

Base Sequence↗

PubMed1999

A fast method to diagnose chromosome and plasmid loss in Saccharomyces cerevisiae strains.

We have developed a simple, fast and reliable method for the analysis of genetic stability in budding yeast strains. The assay relies on our previous finding that cells expressing the green fluorescent protein (GFP) can be detected and counted by flow cytometric analysis (FACS) (Niedenthal et al., 1996). Expression of a gfp-carrying CEN-plasmid in a wild-type strain resulted in the emission of strong fluorescence from 80% of the cell population. Strong fluorescence and presence of the plasmid, determined by the presence of the URA3 genetic marker, was strictly correlated. Expression of this plasmid in 266 yeast strains, each carrying a complete deletion of a novel, non-essential gene identified in the S. cerevisiae sequencing project, pinpointed 12 strains with an increased level of mitotic plasmid loss. Finally we have shown that measurement of mitotic loss of artificial chromosome fragments equipped with the gfp expression cassette can be performed quantitatively using FACS.

Chromosome Segregation↗

PubMed1999

Systematic analysis of S. cerevisiae chromosome VIII genes.

To begin genome-wide functional analysis, we analysed the consequences of deleting each of the 265 genes of chromosome VIII of Saccharomyces cerevisiae. For 33% of the deletion strains a growth phenotype could be detected: 18% of the genes are essential for growth on complete glucose medium, and 15% grow significantly more slowly than the wild-type strain or exhibit a conditional phenotype when incubated under one of 20 different growth conditions. Two-thirds of the mutants that exhibit conditional phenotypes are pleiotropic; about one-third of the mutants exhibit only one phenotype. We also measured the level of expression directed by the promoter of each gene. About half of the promoters direct detectable transcription in rich glucose medium, and most of these exhibited only low or medium activity. Only 1% of the genes are expressed at about the same level as ACT1. The number of active promoters increased to 76% upon growth on a non-fermentable carbon source, and to 93% in minimal glucose medium. The majority of promoters fluctuated in strength, depending on the medium.

DNA Primers↗

PubMed1999

Functional analysis of 150 deletion mutants in Saccharomyces cerevisiae by a systematic approach.

In a systematic approach to the study of Saccharomyces cerevisiae genes of unknown function, 150 deletion mutants were constructed (1 double, 149 single mutants) and phenotypically analysed. Twenty percent of all genes examined were essential. The viable deletion mutants were subjected to 20 different test systems, ranging from high throughput to highly specific test systems. Phenotypes were obtained for two-thirds of the mutants tested. During the course of this investigation, mutants for 26 of the genes were described by others. For 18 of these the reported data were in accordance with our results. Surprisingly, for seven genes, additional, unexpected phenotypes were found in our tests. This suggests that the type of analysis presented here provides a more complete description of gene function.

Cell Differentiation↗

PubMed1999

Ordered assembly of the asymmetrically branched lipid-linked oligosaccharide in the endoplasmic reticulum is ensured by the substrate specificity of the individual glycosyltransferases.

The assembly of the lipid-linked core oligosaccharide Glc3Man9GlcNAc2, the substrate for N-linked glycosylation of proteins in the endoplasmic reticulum (ER), is catalyzed by different glycosyltransferases located at the membrane of the ER. We report on the identification and characterization of the ALG12 locus encoding a novel mannosyltransferase responsible for the addition of the alpha-1,6 mannose to dolichol-linked Man7GlcNAc2. The biosynthesis of the highly branched oligosaccharide follows an ordered pathway which ensures that only completely assembled oligosaccharide is transferred from the lipid anchor to proteins. Using the combination of mutant strains affected in the assembly pathway of lipid-linked oligosaccharides and overexpression of distinct glycosyltransferases, we were able to define the substrate specificities of the transferases that are critical for branching. Our results demonstrate that branched oligosaccharide structures can be specifically recognized by the ER glycosyltransferases. This substrate specificity of the different transferases explains the ordered assembly of the complex structure of lipid-linked Glc3Man9GlcNAc2 in the endoplasmic reticulum.

Base Sequence↗

PubMed1998

Rapid detection of Chlamydia pneumoniae by PCR-enzyme immunoassay.

Chlamydia pneumoniae is an important human respiratory pathogen. Laboratory diagnosis of infection with this organism is difficult. To facilitate the detection of C. pneumoniae by PCR, an enzyme immunoassay (EIA) for analysis of PCR products was developed. Biotin-labeled PCR products generated from the 16S rRNA gene of C. pneumoniae were hybridized to a digoxigenin-labeled probe and then immobilized to streptavidin-coated microtiter plates. Bound PCR product-probe hybrids were detected with antidigoxigenin peroxidase conjugate and a colorimetric substrate. This EIA was as sensitive as Southern blot hybridization for the detection of PCR products and 100 times more sensitive than visualization of PCR products on agarose gels. The diagnostic value of the PCR-EIA in comparison to cell culture was assessed in throat swab specimens from children with respiratory tract infections. C. pneumoniae was isolated from only 1 of 368 specimens tested. In contrast, 15 patient specimens were repeatedly positive for C. pneumoniae by PCR and Southern analysis. All of these 15 specimens were also identified by PCR-EIA. Of the 15 specimens positive by 16S rRNA-based PCR, 13 specimens could be confirmed by omp1-based PCR or direct fluorescent-antibody assay. Results of this study demonstrate that PCR is more sensitive than cell culture for the detection of C. pneumoniae. The EIA described here is a rapid, sensitive, and simple method for detection of amplified C. pneumoniae DNA.

Blotting, Southern↗

PubMed1998

Depolarization of the actin cytoskeleton is a specific phenotype in Saccharomyces cerevisiae.

The yeast actin cytoskeleton is polarized during most of the cell cycle. Certain environmental factors and mutations are associated with depolarization of the actin cytoskeleton. Is depolarization of the actin cytoskeleton a specific response, or is it a nonspecific reaction to harsh conditions or poor metabolism? If depolarization is a nonspecific response, then any mutation that slows growth should induce depolarization. In addition, the number of genes with the depolarization phenotype should constitute a relatively large part of the genome. To address this question, we determined the effect of slow growth on the actin cytoskeleton, and we determined the frequency of mutations that affect the actin cytoskeleton. Eight mutants with slow growth showed no defect in actin polarization, indicating that slow growth alone is not sufficient to cause depolarization. Among 273 viable haploids disrupted for ORFs of chromosome I and VIII and 950 viable haploids with random genome disruptions, none had depolarization of the cytoskeleton. We conclude that depolarization of the actin cytoskeleton is a specific phenotype.

Actins↗

PubMed1997

Mal3, the fission yeast homologue of the human APC-interacting protein EB-1 is required for microtubule integrity and the maintenance of cell form.

Through a screen designed to isolate novel fission yeast genes required for chromosome segregation, we have identified mal3+. The mal3-1 mutation decreased the transmission fidelity of a nonessential minichromosome and altered sensitivity to microtubule-destabilizing drugs. Sequence analysis revealed that the 35-kD Mal3 is a member of an evolutionary conserved protein family. Its human counterpart EB-1 was identified in an interaction screen with the tumour suppressor protein APC. EB-1 was able to substitute for the complete loss of the mal3+ gene product suggesting that the two proteins might have similar functions. Cells containing a mal3 null allele were viable but showed a variety of phenotypes, including impaired control of cell shape. A fusion protein of Mal3 with the Aequorea victoria green fluorescent protein led to in vivo visualization of both cytoplasmic and mitotic microtubule structures indicating association of Mal3 with microtubules. The absence of Mal3 protein led to abnormally short, often faint cytoplasmic microtubules as seen by indirect antitubulin immunofluorescence. While loss of the mal3+ gene product had no gross effect on mitotic spindle morphology, overexpression of mal3+ compromised spindle formation and function and led to severe growth inhibition and abnormal cell morphology. We propose that Mal3 plays a role in regulating the integrity of microtubules possibly by influencing their stability.

Adenomatous Polyposis Coli↗

PubMed1997

The nucleotide sequence of Saccharomyces cerevisiae chromosome XIV and its evolutionary implications.

In 1992 we started assembling an ordered library of cosmid clones from chromosome XIV of the yeast Saccharomyces cerevisiae. At that time, only 49 genes were known to be located on this chromosome and we estimated that 80% to 90% of its genes were yet to be discovered. In 1993, a team of 20 European laboratories began the systematic sequence analysis of chromosome XIV. The completed and intensively checked final sequence of 784,328 base pairs was released in April, 1996. Substantial parts had been published before or had previously been made available on request. The sequence contained 419 known or presumptive protein-coding genes, including two pseudogenes and three retrotransposons, 14 tRNA genes, and three small nuclear RNA genes. For 116 (30%) protein-coding sequences, one or more structural homologues were identified elsewhere in the yeast genome. Half of them belong to duplicated groups of 6-14 loosely linked genes, in most cases with conserved gene order and orientation (relaxed interchromosomal synteny). We have considered the possible evolutionary origins of this unexpected feature of yeast genome organization.

Base Sequence↗

PubMed1997

Sequence analysis of the 33 kb long region between ORC5 and SUI1 from the left arm of chromosome XIV from Saccharomyces cerevisiae.

We have determined the nucleotide sequence of a chromosomal region of 33,016 bp located on the left arm of chromosome XIV from budding yeast between the ORC5 and the SUI1 gene. Subsequent sequence analysis revealed the presence of 18 non-overlapping open reading frames (ORFs) including eight previously identified and sequenced genes (ORC5, ATX1, SIP3, NRD1, RAD50, MPA43, RPA49 and SUI1). Three other ORFs (YNL256w, YNL255c and YNL247w) code for putative proteins with significant homology to proteins from other organisms, while 4 ORFs exhibit only weak homology to known proteins. Three ORFs have no homology with sequences in the databases.

Amino Acid Sequence↗