Horst Domdey

Preparation of high molecular weight RNA

Publisher Summary This chapter presents the procedure for preparation of high molecular weight RNA. High molecular weight RNA can be isolated from haploid or diploid yeast cells as well as from ascospores. The conditions under which the yeast cells are grown (complete or selective media) do not influence the preparation procedure, although higher yields of RNA are generally obtained from cells grown in rich medium. Procedure requires some special precautions owing to the ubiquitous presence of RNA-degrading enzymes. For this reason it is important to wear gloves during the isolation and preparation procedure, and it is essential to separate all chemicals, tubes, and tips used for RNA preparation from commonly used materials. All glassware has to be baked for 2 hr at 200°; commercially available sterile plastic tubes or tips do not have to be specially pretreated, because they seem to be free of fibonucleases. Because the degradation of RNA by RNases is a time-dependent reaction, it is also advisable to work as fast as possible.

Mutations in a Peptidylprolyl-cis/trans-isomerase Gene Lead to a Defect in 3′-End Formation of a Pre-mRNA inSaccharomyces cerevisiae

In a genetic screen aimed at the identification of trans-acting factors involved in mRNA 3′-end processing of budding yeast, we have previously isolated two temperature-sensitive mutants with an apparent defect in the 3′-end formation of a plasmid-derived pre-mRNA. Surprisingly, both mutants were rescued by the essential gene ESS1/PTF1 that encoded a putative peptidylprolyl-cis/trans-isomerase (PPIase) (Hani, J., Stumpf, G., and Domdey, H. (1995) FEBS Lett. 365, 198–202). Such enzymes, which catalyze thecis/trans-interconversion of peptide bonds N-terminal of prolines, are suggested to play a role in protein folding or trafficking. Here we report that Ptf1p shows PPIase activity in vitro, displaying an unusual substrate specificity for peptides with phosphorylated serine and threonine residues preceding proline. Both mutations were found to result in amino acid substitutions of highly conserved residues within the PPIase domain, causing a marked decrease in PPIase activity of the mutant enzymes. Our results are suggestive of a so far unknown involvement of a PPIase in mRNA 3′-end formation in Saccharomyces cerevisiae.

PTF1 encodes an essential protein in Saccharomyces cerevisiae, which shows strong homology with a new putative family of PPIases

Complementation of a temperature sensitive mutant of the yeast Saccharomyces cerevisiae resulted in the isolation of PTF1 (processing/termination factor 1), an essential gene encoding a putative 3′‐end processing or transcription termination factor of pre‐mRNAs. Ptf1p shows significant homology to a newly discovered family of PPIases. This family is characterized by its insensitivity to immunosuppressive drugs and the lack of homology with cyclophilins and FK‐506 binding proteins [Rahfeld et al. (1994) FEBS Lett. 352, 180–184]. Should Ptf1p display PPIase activity, it would be the first characterized, eukaryotic member of this putative family, which is essential for growth.

Dependence of yeast pre-mRNA 3'-end processing on CFT1 : A sequence homolog of the mammalian AAUAAA binding factor

3′-End formation of pre-mRNA in yeast and mammals follows a similar but distinct pathway. In Saccharomyces cerevisiae, the cleavage reaction can be reconstituted by two activities called cleavage factor I and II (CFI and CFII). A CFII component, designated CFT1 (cleavage factor two) was identified by its sequence similarity to the AAUAAA-binding subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF), even though the AAUAAA signal sequence appears to play no role in yeast pre-mRNA 3′ processing. Depletion of a yeast whole-cell extract with antibodies to CFT1 protein abolished cleavage and polyadenylation of pre-mRNAs. Addition of CFII restored cleavage activity, but not polyadenylation. Polyadenylation required the further addition of poly(A) polymerase and polyadenylation factor I, suggesting a close but not necessarily direct association of these two factors with the CFT1 protein.

A recombinant hybrid outer membrane protein for vaccination against Pseudomonas aeruginosa.

Among the numerous targets which can be used for the development of vaccines against Pseudomonas aeruginosa we focused on the outer membrane proteins OprF and OprI. The C-terminal part of OprF from aa 190 to aa 350 was investigated for its conservation and its localization of B-cell epitopes. A hybrid protein which combines the protective epitopes of OprF and OprI was expressed in E. coli and was proven to be highly protective against an intraperitoneal challenge with P. aeruginosa by active immunization of immunocompromised mice as well as by passive immunization of SCID mice with specific antisera. A purification procedure of the N-terminal His-tagged hybrid antigen was established using immobilized-metal-affinity chromatography. To evaluate its safety and immunogenicity the recombinant protein was purified for the immunization of human volunteers. The OprF/OprI hybrid protein is considered to be a candidate for a vaccine against P. aeruginosa.

Safety and immunogenicity of a Pseudomonas aeruginosa outer membrane protein I vaccine in human volunteers

The outer membrane protein I (OprI) of Pseudomonas aeruginosa was expressed in Escherichia coli and purified by Ni2+ chelate-affinity chromatography. After safety and pyrogenicity evaluation in animals, four groups of seven adult human volunteers were vaccinated three times at four week intervals with either 500 micrograms, 200 micrograms, 50 micrograms or 20 micrograms of OprI adsorbed onto Al(OH)3. All vaccinations were well tolerated and no systemic side effects were detected. A significant rise of antibody titers against OprI could be measured in the serum of all volunteers who had received the 500 micrograms, 200 micrograms or 50 micrograms doses. Elevated antibody titers against OprI could still be measured 30 weeks after the final vaccination. Binding of the complement component C1q to the elicited antibodies could be demonstrated, showing the ability of the latter to promote antibody-mediated complement-dependent opsonization.

Assignment of the human organic anion transporting polypeptide (OATP) gene to chromosome 12p12 by fluorescence in situ hybridization

BACKGROUND/AIMS The organic anion transporting polypeptide (OATP) of human liver mediates the basolateral hepatocellular uptake of numerous cholephilic anions and steroidal compounds. The aim of this study was to clone the human OATP gene and to map its chromosomal localization by fluorescence in situ hybridization. METHODS A polymerase chain reaction-amplified fragment of the human OATP gene was used to isolate a genomic OATP clone from a P1-derived artificial chromosome human genomic library. Human metaphase chromosomes were hybridized with digoxigenin-labeled DNA from the genomic OATP clone and incubated in fluoresceinated antidigoxigenin antibodies for in situ detection of specific hybridization signals. RESULTS Sequence analysis revealed that the isolated P1-derived artificial chromosome clone contained a large portion of the human OATP gene. Fluorescence in situ hybridization of human chromosomes with the genomic OATP clone resulted in the specific labeling of the OATP gene on the short arm of chromosome 12 at band 12p12. CONCLUSIONS Mapping of a genomic OATP clone to chromosome 12p12 represents a first step towards the molecular characterization of the human OATP gene. While no liver disease has so far been associated with cytogenetic abnormalities of the short arm of chromosome 12, the genomic OATP sequence provides the basis for studies on gene structure and on the tissue-specific regulation of OATP gene expression.

Structure and Expression of the C3 Gene

To map the multiple interactive sites on the C3 polypeptide, it is advantageous to combine the approaches of protein chemistry, nucleic acid technology, and molecular biology. This review summarizes the currently known molecular properties of mouse liver C3 mRNA, cloned C3 cDNA, and genomic DNA. Original data communicated have specified the amino acid sequence of the 215 amino-terminal residues of mature mouse C3 beta. Southern blot analysis of liver DNA indicated that the mouse genome contains only one type of C3 gene, that murine and human C3 sequences strongly cross-hybridize, and that the human C3 gene is not somatically rearranged. Included are descriptions of the first human C3 genomic DNA clones, their preparation, and their use to map the human C3 gene to chromosome 19 in linkage with the myotonic dystrophy (DM) locus. After a brief survey of reports describing inherited human C3 deficiencies, we discuss a Dutch family and their three members with total homozygous C3 deficiency who were the subjects of a recent publication. The restricted synthesis of C3 in major and minor producer tissues is discussed and it is proposed that the C3 gene provides a good model system for studying the molecular basis of tissue-specific gene expression. Data are presented documenting the production of C3 in two established mouse macrophage-like cell lines and two rat hepatoma cell lines in tissue cultures. A short account covers the extensive literature on regulation of C3 serum concentrations in acute and chronic inflammation and the very incomplete picture that presently depicts hormonal regulation of C3 synthesis. The final experiment reported demonstrates that nucleic acid hybridization with cloned cDNA probes is a sensitive assay for quantitative determinations of C3 mRNA. With the help of cloned cDNA and genomic DNA, researchers can address questions concerning the functional topography of the C3 polypeptide, the genes structure, and the molecular nature of inherited C3 deficiencies in humans.

The intron of the yeast actin gene contains the promoter for an antisense RNA

SummaryUsing Northern blot analysis we have detected an approximately 840 nucleotide-long RNA which is complementary to the 5′ leader sequence and the first ten nucleotides of the coding sequence of the yeast actin (ACT1) messenger RNA. We have determined two transcription start sites for this actin antisense RNA (ASR1), both within the ACT1 intron, at about 80 and 90 nucleotides downstream from the 5′ splice site. Analysis of a cDNA clone showed that this RNA species overlaps the entire trailer sequence and approximately 20 nucleotides of the coding sequence of the nearby yeast YPT1 gene.

The beta-tubulin genes of Drosophila auraria are arranged in a cluster.

When the β1-, β2- and β3-tubulin-specific DNAs fromDrosophila melanogaster were used as probes to recognize tubulin-specific sequences in the chromosomes ofDrosophila auraria, they were found to hybridize to the same polytene band in region 32C of the 2L polytene chromosome. Three overlapping clones were isolated from a λEMBL 3 genomic library ofD. auraria, and they all contain β-tubulin-specific sequences based on hybridization and partial-sequencing experiments of subcloned fragments. These clones hybridize in situ to the same polytene chromosome band in region 32C and they represent an approximately 35-kb fragment of genomic DNA.