cDNA-derived primary structure of the glycoprotein component of canine microsomal signal peptidase complex

Canine microsomal signal peptidase activity has been shown previously to co-migrate as an apparent complex of six polypeptides with molecular masses of 25, 23, 22, 21, 18, and 12 kDa. The 22- and 23-kDa species are differentially glycosylated forms of the same protein, designated SPC 22/23. The amino acid sequence of SPC 22/23 was deduced from cDNA clones. The protein is synthesized without a cleavable amino-terminal signal sequence and contains a single site for N-linked glycosylation. SPC 22/23 appears to be anchored to the rough endoplasmic reticulum membrane by a single hydrophobic segment near its amino terminus, with the remainder of the protein positioned on the lumenal side of the membrane. The amino acid sequence of SPC 22/23 shares homology with tryptic peptides derived from the hen oviduct signal peptidase glycoprotein, one of two possible proteins required for signal peptide processing in the avian system (Baker, R.K., and Lively, M.O. (1987) Biochemistry 26, 8561-8567). Therefore, the complete amino acid sequence of SPC 22/23 presented in this report corresponds to one of two possible proteins required for signal peptide processing in higher eukaryotic cells.     

Specific binding sites for monomeric and aggregated beta 2-microglobulin on surface of groups A, B, C, and G streptococci

Human beta 2-microglobulin (beta 2-m) was isolated from urine samples of patients with tubular dysfunctions and aggregated with glutaraldehyde. Four aggregates with molecular weights of 800,000, 480,000, 260,000, and 60,000 were separated by filtration on Sephacryl S-300. The aggregates and monomeric beta 2-m (11,800 MW) were subsequently labeled with 125I and tested for binding to streptococci. Group A streptococci bound only aggregated beta 2-m with a mean binding of 44.5%. Most of the group G streptococci, on the other hand, bound only monomeric beta 2-m with a mean binding of 58%. Among group B streptococci the serotypes with protein antigens interacted mainly with monomeric beta 2-m and those without protein antigens preferentially with aggregated beta 2-m. Nontypable group B streptococcal serotypes did not bind monomeric or aggregated beta 2-m. Of the streptococci belonging to group C, S. equisimilis reacted with monomeric beta 2-m and S. dysgalactiae with aggregated beta 2-m. S. equi did not interact with monomeric beta 2-m or aggregated beta 2-m. Bindings of monomeric beta 2-m and aggregated beta 2-m were saturable and could be inhibited by the respective unlabeled forms of beta 2-m. Fibrinogen, fibronectin, alpha 2-macroglobulin, haptoglobin, or immunoglobulin G did not inhibit the binding of either form of beta 2-m. The binding sites for monomeric beta 2-m were more susceptible to trypsin than those for aggregated beta 2-m. Treatment of streptococci with pronase destroyed their binding activities for monomeric and aggregated beta 2-m. Both monomeric beta 2-m and aggregated beta 2-m binding sites were sensitive to heat. The Scatchard plots of monomeric beta 2-m and aggregated beta 2-m were linear with Kd of 1.29 X 10(-9) M and 1.9 X 10(-9) M respectively. The number of binding sites per bacterium were estimated to be 81,000 for monomeric beta 2-m and 1,210 for aggregated beta 2-m.     

Isolation and characterization of a large,neurite-associated glycoconjugate from neuroblastoma cells

A high molecular weight glycoconjugate has been isolated from neurite-producing neuronal tumor cells in culture and has been designated as I(0) based on its elution characteristics in gel filtration chromatography. This molecule cannot be found in a variety of nonneuronal cells. I(0) is found in the substratum-attached material or cell fraction of neurite-producing neuroblastoma cells, depending upon culture conditions. It is found in the substratum-bound fraction of B104 rat neuroblastoma cells during serum starvation and in the EGTA-detached cell fraction of B104 cells grown in chemically defined N2 medium. It occurs only in the cell fraction of the human neuroblastoma line Platt. Examination of behavioral variants of the B104 rat line further strengthens the association of I(0) with neurite production; the constitutive neurite-producing E(R)B9 variant contains I(0) while the non-neurite-producing E(R)A11 variant does not. I(0) is large, eluting in the void volume of sepharose-CL2B columns. Radioiodination of intact cells with lactoperoxidase shows I(0) to be a cell surface component. Metabolic radiolabeling studies show that it contains a high proportion of polysaccharide to protein, does not contain mannose, and is unsulfated. Alkaline borohydride reduction release two size classes of large polysaccharide chain. The alkaline reduction results, along with the mannose incorporation studies, show the presence of O-glycosidic linkages and few, if any, N-linkages. Resistance to nitrous acid deamination, insensitivity to glycosaminoglycan lyases, and the absence of sulfation, indicate that I(0) does not contain the glycosaminoglycans hyaluronic acid, chondroitin-, dermatan-, or heparin- sulfates. Affinity column chromatography reveals high binding affinity of I(0) to polyornithine and no binding to gelatin (collagen) or the glycosaminoglycans hyaluronate and heparin. These studies describe a unique high molecular weight glycoconjugate on the surface of neurite-producing neuroblastoma cell lines from two species.     

ISOLATION AND CHARACTERIZATION OF DEOXYRIBONUCLEIC ACID FROM A STRAIN OF STAPHYLOCOCCUS AUREUS.

Blobel, Hans (University of Wisconsin, Madison). Isolation and characterization of deoxyribonucleic acid from a strain of Staphylococcus aureus. J. Bacteriol. 82:425-429. 1961.-Highly polymerized deoxyribonucleic acid was extracted from washed cells of Staphylococcus aureus with a mixture of equal parts of phenol and 2 m NaCl at pH 7.4. The aqueous phase was treated twice with phenol and the deoxyribonucleic acid precipitated with an equal volume of 2-ethoxyethanol. Residual ribonucleic acid was removed by treatment with ribonuclease and subsequent dialysis. Deoxyribonucleic acid was reprecipitated with 2-ethoxyethanol. The final product contained less than 1% protein. The deoxyribonucleic acid obtained from S. aureus strain S(44) had a (adenosine + thymine)/(guanine + cytosine) base ratio of 1.98. Intrinsic viscosity in 0.15 m NaCl + 0.015 m sodium citrate was approximately 76 dl/g. The sedimentation coefficient, S(20)w, was close to 25 S.     

Yeast nuclear envelope proteins cross react with an antibody against mammalian pore complex proteins

We have used a monoclonal antibody raised against rat liver nuclear proteins to study two cross-reactive proteins in the yeast nucleus. In rat liver, this monoclonal antibody, mAb 414, binds to nuclear pore complex proteins, including one of molecular weight 62,000 (Davis, L. I., and G. Blobel. 1987. Proc. Natl. Acad. Sci. USA. 84:7552-7556). In yeast, mAb 414 cross reacts by immunoblotting with two proteins that have apparent molecular weights of 110,000 and 95,000, and are termed p110 and p95, respectively. Examination of subcellular fractions by immunoblotting shows that both p110 and p95 are located exclusively in the nuclear fraction. The mAb 414 immunoprecipitates several proteins from a crude yeast cell extract, including p110, p95, and a approximately 55-kD protein. Immunoprecipitation from subcellular fractions yields only p110 and p95 from purified nuclei, whereas the approximately 55-kD protein is immunoprecipitated from the soluble fraction. Digestion of purified nuclei with DNase to produce nuclear envelopes releases some of p110, but the majority of p110 is solubilized only after treatment of envelopes with 1 M NaCl. Immunofluorescence localization using yeast cells and isolated nuclei shows a punctate and patchy staining pattern of the nucleus. Confocal laser scanning immunofluorescence microscopy resolves the punctate and patchy staining pattern better and shows regions of fluorescence at the nuclear envelope. Postembedding immunogold electron microscopy using purified nuclei and mAb 414 shows colloidal gold decoration of the yeast nuclear envelope, but resolves pore complexes too poorly to achieve further ultrastructural localization. Immunogold labeling of nuclei followed by embedding suggests decoration of pore complexes. Thus, p110 and/or p95 are localized to the nuclear envelope in yeast, and may be components of the nuclear pore complex.     

Plasmid-encoded resistance to macrolides and lincosamides in Staphylococcus hyicus

S chwarz , S., W egener , H. & B lobel , H. 1990. Plasmid-encoded resistance to macrolides and lincosamides in Staphylococcus hyicus. Journal of Applied Bacteriology 69 , 845–849.
A small plasmid of 2–35 kb, isolated from a porcine Staphylococcus fcyicus-culture, was found to be responsible for constitutive resistance to macrolide/lincosamide antibiotics. This plasmid-encoded property could be established by interspecific transformation experiments. The plasmid from porcine Staph. hyicus was designated as pSE2. It differed on the basis of its restriction map from the macrolid/lincosamid resistance (ML^R-)-plasmids of other staphylococcal species from infections of humans. Furthermore, the pSE2 plasmid encoded two proteins of approximately 20.5 and 30 kDa.     

Relative apparent synapomorphy analysis (RASA). I: The statistical measurement of phylogenetic signal

We have developed a new approach to the measurement of phylogenetic signal in character state matrices called relative apparent synapomorphy analysis (RASA). RASA provides a deterministic, statistical measure of natural cladistic hierarchy (phylogenetic signal) in character state matrices. The method works by determining whether a measure of the rate of increase of cladistic similarity among pairs of taxa as a function of phenetic similarity is greater than a null equiprobable rate of increase. Our investigation of the utility and limitations of RASA using simulated and bacteriophage T7 data sets indicates that the method has numerous advantages over existing measures of signal. A first advantage is computational efficiency. A second advantage is that RASA employs known methods of statistical inference, providing measurable sensitivity and power. The performance of RASA is examined under various conditions of branching evolution as the number of characters, character states per character, and mutations per branch length are varied. RASA appears to provide an unbiased and reliable measure of phylogenetic signal, and the general approach promises to be useful in the development of new techniques that should increase the rigor and reliability of phylogenetic estimates.      

Molecular evolution of voltage-sensitive ion channel genes: on the origins of electrical excitability

We have analyzed nucleic acid and amino acid sequence alignments of a variety of voltage-sensitive ion channels, using several methods for phylogenetic tree reconstruction. Ancient duplications within this family gave rise to three distantly related groups, one consisting of the Na+ and Ca++ channels, another the K+ channels, and a third including the cyclic nucleotide-binding channels. A series of gene duplications produced at least seven mammalian homologues of the Drosophila Shaker K+ channel; clones of only three of these genes are available from all three mammalian species examined (mouse, rat, and human), pointing to specific genes that have yet to be recovered in one or another of these species. The Shaw-related K+ channels and the Na+ channel family have also undergone considerable expansion in mammals, relative to flies. These expansions presumably reflect the needs of the high degree of physiological and neuronal complexity of mammals. Analysis of the separate domains of the four-domain channels (Ca++ and Na+) supports their having evolved by two sequential gene duplications and implies the historical existence of a functional two-domain channel.      

mtDNA diversity in rhesus monkeys reveals overestimates of divergence time and paraphyly with neighboring species

Reconstructions of the human-African great ape phylogeny by using mitochondrial DNA (mtDNA) have been subject to considerable debate. One confounding factor may be the lack of data on intraspecific variation. To test this hypothesis, we examined the effect of intraspecific mtDNA diversity on the phylogenetic reconstruction of another Plio- Pleistocene radiation of higher primates, the fascicularis group of macaque (Macaca) monkey species. Fifteen endonucleases were used to identify 10 haplotypes of 40-47 restriction sites in M. mulatta, which were compared with similar data for the other members of this species group. Interpopulational, intraspecific mtDNA diversity was large (0.5%- 4.5%), and estimates of divergence time and branching order incorporating this variation were substantially different from those based on single representatives of each species. We conclude that intraspecific mtDNA diversity is substantial in at least some primate species. Consequently, without prior information on the extent of genetic diversity within a particular species, intraspecific variation must be assessed and accounted for when reconstructing primate phylogenies. Further, we question the reliability of hominoid mtDNA phylogenies, based as they are on one or a few representatives of each species, in an already depauperate superfamily of primates.