Repeated sequence elements in the high molecular weight basic nuclear proteins from the winter flounder

In maturing sperm of the winter flounder, histones are not replaced by protamines but instead joined by a group of high molecular weight basic nuclear proteins. Despite their large size and number of components, these proteins were reduced to a relatively simple set of peptides by a "limit" digestion with endoprotease Lys-C. Nine of these peptides, that together account for half of the mass of the digest, were purified by two rounds of chromatography on a C18 reverse-phase high pressure liquid chromatographic column and analysed by sequential Edman degradation. Their sequences can be divided into two homology groups. Seven of the peptides contain all or part of a dodecapeptide consensus sequence, NH2-Ser-Pro-Met-Arg-Ser-Arg-Ser-Pro-Ser-Arg-Ser-Lys-COOH, which appears to be tandemly repeated. This dodecapeptide contains a previously recognized consensus phosphorylation sequence, NH2-Arg-Ser-Arg-Ser-Pro-COOH, in which both serines are phosphorylated during the early stages of spermiogenesis. The other homology group has the sequence NH2-Arg-Arg-Val-X-X-Pro-Lys-COOH, where X-X is either Gln-Thr or Pro-Ser. The dodecapeptide and heptapeptide sequences form at least 35 and 11%, respectively, of the high molecular weight basic nuclear proteins and are, therefore, repeated many times over in these proteins. A search for identical or homologous sequences within the Protein Sequence Database indicated that they are unique. The closest matches were to protamines and some viral DNA-binding proteins.     

Abnormal subcellular distribution of β-glucuronidase in mice with a genetic alteration in enzyme structure

Liver -glucuronidase is structurally altered in inbred strain PAC so that a peptide subunit with a more basic isoelectric point, GUS-SN, is produced. This allele of -glucuronidase was transferred to strain C57BL/6J by 12 backcross matings to form the congenic line B6 · PAC-Gus ⁿ. Liver -glucuronidase activity was halved in males of the congenic strain compared to normal males. The lowered activity was specifically accounted for by a decrease in the lysosomal component. There was no alteration in the concentration of microsomal activity. This alteration in the subcellular distribution of -glucuronidase in Gus ⁿ/Gus ⁿ mice was confirmed by two independent gel electrophoretic systems which separate microsomal and lysosomal components. -Glucuronidase activity was likewise approximately halved in mutant spleen, lung, and brain, organs which contain exclusively or predominantly lysosomal -glucuronidase. The loss of liver lysosomal -glucuronidase activity was shown by immunotitration to be due to a decrease in the number of -glucuronidase molecules in lysosomes of the congenic strain. The Gus ⁿ structural alteration likely causes the lowered lysosomal -glucuronidase activity since the two traits remain in congenic animals. Heterozygous Gus ⁿ/Gus ^b animals had intermediate levels of liver -glucuronidase. Also, the effect was specific, in that three other lysosomal enzymes were not reproducibly lower in Gus ⁿ/Gus ⁿ mice. Gus ⁿ is, therefore, an unusual example of a mutation which causes a change in the subcellular distribution of a two-site enzyme.This work was supported by National Institutes of Health Grants GM-33559 and GM-33160 and National Science Foundation Grant PCM-8215808.     

Occurrence of bacterivory in Cryptomonas,a common freshwater phytoplankter

Summary Bacterivory was detected by incorporation of 0.57 m diameter, fluorescent polystyrene beads and fluorescently labeled bacteria (FLB) in two cultured species of Cryptomonas (C. ovata and C. erosa), and a population of Cryptomonas sp in a humic, mesotrophic lake. Rates of ingestion and clearance were very low, and similar for the cultures and the in situ population. The in situ population incorporated 0.7–1.7 bacteria cell^-1 h^-1, thereby ingesting 0.3%–2.0% of the total bacterial numbers present in the water per day, and receiving less than 2% of its carbon content per day through bacterivory. Thus, bacterivory by Cryptomonas was quantitatively important neither as a sink for bacterial biomass, nor as a carbon source for the algal cells. Possibly, it served in the uptake of essential nutrients.     

Mutations in the signal sequence of prepro-alpha-factor inhibit both translocation into the endoplasmic reticulum and processing by signal peptidase in yeast cells.

The effects of five single-amino-acid substitution mutations within the signal sequence of yeast prepro-alpha-factor were tested in yeast cells. After short pulse-labelings, virtually all of the alpha-factor precursor proteins from a wild-type gene were glycosylated and processed by signal peptidase. In contrast, the signal sequence mutations resulted in the accumulation of mostly unglycosylated prepro-alpha-factor after a short labeling interval, indicating a defect in translocation of the protein into the endoplasmic reticulum. Confirming this interpretation, unglycosylated mutant prepro-alpha-factor in cell extracts was sensitive to proteinase K and therefore in a cytosolic location. The signal sequence mutations reduced the rate of translocation into the endoplasmic reticulum by as much as 25-fold or more. In at least one case, mutant prepro-alpha-factor molecules were translocated almost entirely posttranslationally. Four of the five mutations also reduced the rate of proteolytic processing by signal peptidase in vivo, even though the signal peptide alterations are not located near the cleavage site. This study demonstrates that a single-amino-acid substitution mutation within a eucaryotic signal peptide can affect both translocation and proteolytic processing in vivo and may indicate that the recognition sequences for translocation and processing overlap within the signal peptide.     

Characterization of D2 receptors and dopamine levels in the thalamus of the rat.

We kinetically characterized D2 receptors in thalami pooled from a group of Sprague-Dawley rats and then determined thalamic levels of dopamine (DA), homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC), and norepinephrine (NE) in relation to a measure of thalamic DA D2 receptor densities in another group of rats. The equilibrium dissociation constant (kd) was estimated as 0.1 nM by three independent methods, while the Bmax for thalamic D2 receptors was found to be 6.4 fmol/mg p using 3H-spiperone as ligand and ketanserin to occlude 5HT2 binding. Kinetic constants were in agreement with previously reported kinetic data from rodent caudate-putamen. This suggests that thalamic D2 receptors are similar to D2 receptors from other brain areas. Mean thalamic levels of DA (22.6 ng/mg p), DOPAC (1.19 ng/mg p) and HVA (0.31 ng/mg p) concur with previous reports of a sparse distribution of thalamic DA neurons. D2 receptor densities were positively correlated with DA metabolites DOPAC (P less than .05; r = 0.423) and HVA (P less than .05; r = 0.368), but not DA or NE. These results establish fundamental characteristics of thalamic DA neurotransmission to assist in the investigation of behavioral pharmacology of this area.     

Evaluation of Diverse Antisera, Conjugates, and Support Media for Detecting Bradyrhizobium japonicum by Indirect Enzyme-Linked Immunosorbent Assay

We evaluated three antisera and four enzyme conjugates for the detection of Bradyrhizobium japonicum by an indirect enzyme-linked immunosorbent assay in microtiter plates. Nitrocellulose membrane sheets were then evaluated as an alternative support medium by using some combinations. Partially purified immunoglobulin G (IgG) or unpurified antisera to strain USDA 110 raised in rabbits, goats, or sheep was reacted in microtiter plates with alkaline phosphatase conjugated to protein A, goat anti-rabbit (GAR), sheep anti-rabbit (SAR), or rabbit anti-goat (RAG) IgG. Cultures or nodules containing homologous rhizobia were detected with equal sensitivity when protein A, GAR, or SAR was reacted with 5 μg of protein IgG per ml or a 1:800 titer of antisera from rabbits, but not goats or sheep. RAG reacted with IgG or antisera from goats or sheep. The detection limit was 2 × 10⁵ rhizobia per well. Rhizobia were spotted on nitrocellulose sheets as an alternative support medium, followed by soaking in 5 μg of protein per ml as IgG and 1:4,000 dilutions of protein A or GAR conjugate. Rhizobia in serogroup 110 were detected with the dye combination Nitro Blue Tetrazolium-5-bromo-4-chloro-3-indolyl phosphate (NBT-BCIP), and rhizobia in serogroup 122 were detected with fast red-naphthol phosphate (FR-NP). At the conclusion of the 5-h assay, purple (NBT-BCIP) or red (FR-NP) spots were visible in positive reactions. The sensitivity of detection was about 1,000 rhizobial cells or 3 μg of nodules tissue.