Ruedi Aebersold

A cellular gene encodes scrapie PrP 27-30 protein.

A clone encoding PrP 27-30, the major protein in purified preparations of scrapie agent, was selected from a scrapie-infected hamster brain cDNA library by oligonucleotide probes corresponding to the N terminus of the protein. Southern blotting with PrP cDNA revealed a single gene with the same restriction patterns in normal and scrapie-infected brain DNA. A single PrP-related gene was also detected in murine and human DNA. PrP-related mRNA was found at similar levels in normal and scrapie-infected hamster brain, as well as in many other normal tissues. Using antisera against PrP 27-30, a PrP-related protein was detected in crude extracts of infected brain and to a lesser extent in extracts of normal brain. Proteinase K digestion yielded PrP 27-30 in infected brain extract, but completely degraded the PrP-related protein in normal brain extract. No PrP-related nucleic acids were found in purified preparations of scrapie prions, indicating that PrP 27-30 is not encoded by a nucleic acid carried within the infectious particles.

Analysis of dilute peptide samples by capillary zone electrophoresis

We report a method for the analysis of dilute peptide solutions by capillary zone electrophoresis. The procedure is based on an electrophoretic concentration step of the applied peptide solution in the capillary (stacking) prior to separation, thus allowing the application of increased sample volumes without a breakdown in resolution. Given a constant configuration of the hardware, the method permits the analysis of peptide solutions of an at least 5 times lower concentration than previously possible. The method was applied to the direct analysis of peptide samples separated by narrow-bore reversed-phase high-performance liquid chromatography for high-sensitivity peptide-sequence analysis.

Characterization of a spinach psbS cDNA encoding the 22 kDa protein of photosystem II

An intrinsic 22 kDa polypeptide is found associated with the oxygen‐evolving photesytem II (PSII) core complex in all green plants and cyanobacteria so far examined, although it does not appear to be required for oxygen evolution. Amino acid sequence information obtained from the purified 22 kDa protein was used to construct a probe that was employed to isolate a full‐length cDNA clone encoding the 274‐residue precursor of the 22 kDa protein. Hydropathy plot analysis predicts the existence of four membrane‐spanning helices in the mature protein. The two halves of the approximattely 200‐residue mature protein show high sequence similarity to each other, suggesting that the psbS gene arose from an internal gene duplication. The 22 kDa protein has some sequence similarity to chlorophyll a/b‐binding, proteins.

The myelin proteins of the shark brain are similar to the myelin proteins of the mammalian peripheral nervous system

SummaryThe two major structural proteins in the shark CNS are similar to the structural proteins, Po and myelin basic protein (MBP), found in the mammalian peripheral nervous system (PNS). Shark Po is 46% similar to its mammalian counterpart. The extracellular domain of shark Po also appears to be organized as an immunoglobulin-like domain that mediates homotypic interactions. The intracellular domain of shark Po also is very basic and may play a role in myelin condensation analogous to that of MBP. Shark MBP is 44% similar to mammalian MBP. Both MBPs show conserved interspersed regions and are present in multiple forms that arise by alternative splicing of a single transcript. These structural analyses indicate that the complexities seen in mammalian myelin arose early during vertebrate evolution.

Molecular cloning and expression of the gene encoding the kinetoplast-associated type II DNA topoisomerase of Crithidia fasciculata.

A type II DNA topoisomerase, topoIImt, was shown previously to be associated with the kinetoplast DNA of the trypanosomatid Crithidia fasciculata. The gene encoding this kinetoplast-associated topoisomerase has been cloned by immunological screening of a Crithidia genomic expression library with monoclonal antibodies raised against the purified enzyme. The gene CfaTOP2 is a single copy gene and is expressed as a 4.8-kb polyadenylated transcript. The nucleotide sequence of CfaTOP2 has been determined and encodes a predicted polypeptide of 1239 amino acids with a molecular mass of 138,445. The identification of the cloned gene is supported by immunoblot analysis of the beta-galactosidase-CfaTOP2 fusion protein expressed in Escherichia coli and by analysis of tryptic peptide sequences derived from purified topoIImt. CfaTOP2 shares significant homology with nuclear type II DNA topoisomerases of other eukaryotes suggesting that in Crithidia both nuclear and mitochondrial forms of topoisomerase II are encoded by the same gene.

Chlorophyll a/b binding (CAB) polypeptides of CP29, the internal chlorophyll a/b complex of PSII: characterization of the tomato gene encoding the 26 kDa (type 1) polypeptide, and evidence for a second CP29 polypeptide

SummaryCP29, the core chlorophyll a/b (CAB) antenna complex of Photosystem II (PSII), has two nuclearencoded polypeptides of approximately 26 and 28 kDa in tomato (Lycopersicon esculentum). Cab9, the gene for the Type 1 (26 kDa) CP29 polypeptide was cloned by immunoscreening a tomato leaf cDNA library. Its identity was confirmed by sequencing tryptic peptides from the mature protein. Cab9 is a single-copy gene with five introns, the highest number found in a CAB protein. In vitro transcription-translation gave a 31 kDa precursor which was cleaved to about 26 kDa after import into isolated tomato chloroplasts. The Cab9 polypeptide has the two highly conserved regions common to all CAB polypeptides, which define the members of this extended gene family. Outside of the conserved regions, it is only slightly more closely related to other PSII CABs than to PSI CABs. Sequence analysis of tryptic peptides from the Type II (28 kDa) CP29 polypeptide showed that it is also a member of the CAB family and is very similar or identical to the CP29 polypeptide previously isolated from spinach. All members of the CAB family have absolutely conserved His, Gln and Asn residues which could ligate the Mg atoms of the chlorophylls, and a number of conserved Asp, Glu, Lys and Arg residues which could form H-bonds to the polar groups on the porphyrin rings. The two conserved regions comprise the first and third predicted trans-membrane helices and the stroma-exposed segments preceding them.

High-yield recovery of electroblotted proteins and cleavage fragments from a cationic polyvinylidene fluoride-based membrane

In this report we describe the use of a novel, experimental, polyvinylidene fluoride-based membrane with a cationic surface for the isolation by electroblotting of small amounts of proteins separated by gel electrophoresis for further characterization by protein fragmentation for internal sequence analysis. The membrane is characterized by a surface that mediates primarily ionic protein/membrane interactions and that allows the recovery of adsorbed proteins at high yields under relatively mild conditions. In electroblotting experiments, the novel membrane has a binding capacity that is at least equivalent to that of standard polyvinylidene fluoride membranes and is compatible with both chemical and enzymatic fragmentation of blotted proteins in situ. Intact electroblotted proteins, or fragments thereof, were eluted at high yields. Further structural analysis is demonstrated using reverse-phase high-performance liquid chromatography or gel electrophoresis to separate cleavage fragments for either pulsed-liquid- or solid-phase automated sequence analysis.

Identification of the sites in myelin basic protein that are phosphorylated by meiosis-activated protein kinase p44mpk

Myelin basic protein serves as a convenient substrate for detection of a 44 kDa protein‐serine/threonine kinase (p44 mpk ) that is activated near the time of germinal vesicle breakdown in maturing echinoderm and amphibian oocytes. In vitro phosphorylation by purified p44 mpk from sea star oocytes was primarily on threonine residues on a single tryptic peptide of bovine brain myelin basic protein. Amino acid composition analysis of the isolated posphopeptide revealed that it was rich in proline residues. Automated solid‐phase sequencing by Edman degradation identified the major site as Thr‐97 in the sequence NIVTPRTPPPSQGK, which corresponds to residues 91–104 in bovine brain myelin basic protein. Thr‐94 was also phosphorylated by p44 mpk to a very minor extent.

Partial N-terminal amino acid sequences of three nonstructural proteins of two flaviviruses

Partial N-terminal amino acid sequences for the three largest nonstructural proteins of two flaviviruses, yellow fever virus and St. Louis encephalitis virus, have been obtained. The determined sequences of these proteins exhibit significant amino acid sequence homology, and allow the positioning of these three nonstructural proteins in the polyprotein sequence deduced from the nucleotide sequence of yellow fever virus (C. M. Rice, E. M. Lenches, S. R. Eddy, S. J. Shin, R. L. Sheets, and J. H. Strauss, 1985, Science 229, 726-733.) The deduced start points support the hypothesis that the N terminus of nonstructural glycoprotein NS1 results from cleavage by signalase, whereas the N termini of NS3 and NS5 result from cleavages following double basic residues that are flanked by amino acids with short side chains.

The insulin-elicited 160 kDa phosphotyrosine protein in mouse adipocytes is an insulin receptor substrate 1: identification by cloning.

Insulin elicits the tyrosine phosphorylation of one or more proteins of 160-185 kDa in many cell types. Peptide sequences, obtained from this protein purified from mouse 3T3-L1 adipocytes (pp160), were used as the basis for cloning its cDNA, pp160 is highly homologous to the insulin receptor substrate 1, previously cloned from rat liver. Thus, this component of the insulin signaling pathway is the same in adipocytes and in liver.