Isolation and regional localization of a large collection (2,000) of single-copy DNA fragments on human chromosome 3 for mapping and cloning tumor suppressor genes

Summary A collection of 2,000 lambda phage-carrying human single-copy inserts (> 700 bp) were isolated from two chromosome-3 flow-sorted libraries. The single-copy DNA fragments were first sorted into 3p and 3q locations and about 700 3p fragments were regionally mapped using a deletion mapping panel comprised of two humanhamster and two-human-mouse cell hybrids, each containing a chromosome 3 with different deletions in the short arm. The hybrids were extensively mapped with a set of standard 3p markers physically localized or ordered by linkage. The deletion mapping panel divided the short arm into five distinct subregions (A-E). The 3p fragments were distributed on 3p regions as follows: region A, 26%; B, 31%; C, 4%; D, 4% and E, 35%. We screened 300 single-copy DNA fragments from the distal part of 3p (regions A and B) with ten restriction endonucleases for their ability to detect restriction fragment length polymorphisms (RFLPs). Of these fragments 110 (36%) were found to detect useful RFLPs: 35% detected polymorphisms with frequency of heterozygosity of 40% or higher, and 25% with frequency of 30% or higher. All polymorphisms originated from single loci and most of them were of the base pair substitution type. These RFLP markers make it possible to construct a fine linkage map that will span the distal part of chromosome 3p and encompasses the von Hippel-Lindau disease locus. The large number of single-copy fragments (2,000) spaced every 100–150 kb on chromosome 3 will make a significant contribution to mapping and sequencing the entire chromosome 3. The 300 conserved chromosome 3 probes will increase the existing knowledge of man-mouse homologies.     

Spawning behaviour ofCheimerius nufar in captivity

Synopsis The santerCheimerius nufar is widespread in tropical and subtropical waters of the western Indian Ocean and forms an important component of linefish catches along the east coast of southern Africa. Observations of spawning behaviour in captivity have revealed that spawning occurs during spring over a period of four months. Mating takes place at sunrise and may continue for up to 105 min (mean duration = 60 min). During spawning males become dark with prominent white markings. They become very aggressive and set up territories. Females remain a uniform silvery-pink. Mating occurs between males and females of similar sizes, culminating in egg and sperm release near the surface. Individual fish spawned up to 14 times during a morning. Streaking occurred throughout the season, with a second male joining a spawning pair and releasing gametes simultaneously. Slinger,Chrysoblephus puniceus, a dominant fish on offshore reefs in the same region, interfered with spawning throughout the season and were observed to eat eggs when they were released. The spawning strategy ofC. nufar is similar to protogynous species in several respects, indicating that this functional gonochorist may not conform to current theoretical predictions.     

The association between amount of red muscle and mobility in fishes: A statistical evaluation

Synopsis It is commonly accepted that more active fishes have a greater proportion of red muscle in their trunk musculature than do less active fishes. Further, the proportion of red muscle has been used to classify fish species into functional groups reflecting different activity patterns. Nevertheless, existing measures of both red muscle and mobility have several limitations, and the relationship between these parameters has never been evaluated quantitatively. Using data from the literature, we demonstrate a positive, statistical association between the proportion of red muscle in the caudal peduncle of marine fishes and a qualitative measure of mobility (categorization as sedentary vs. mobile based on natural-history accounts). Analyses of the frequency distribution of the proportion of red muscle also provide evidence for two subdistributions. However, this bimodality does not correspond with sedentary vs. mobile or sit-and-wait vs. active search dichotomies.     

A radiochemical assay for a NADP+-specific gamma-glutamate semialdehyde dehydrogenase extracted from mitochondrial membrane of rat intestinal epithelial cells

A radiochemical assay has been developed for a NADP+-specific gamma-glutamate semialdehyde dehydrogenase from rat intestinal epithelial cells. The spectrophotometric assay utilized to measure the enzyme in bacterial cell homogenates is not sensitive enough for homogenates from rat mitochondria, which require an assay that can measure as little as 0.5 nmol NADPH formed/min/ml extract. The assay described here is sensitive to 0.1 nmol product formed/min/ml of extract and employs the use of [3H]pyrroline 5-carboxylate which is phosphorylated and oxidized by the enzyme to gamma-[3H]glutamyl phosphate, a product that decomposes to [3H]pyrrolidone 5-carboxylate. The latter product is separated from the substrate by ion-exchange chromatography. In order to correct for any product loss during separation by ion-exchange [14C]pyrrolidone 5-carboxylate is added as an internal standard to the deproteinized assay mixture. Under the assay conditions described mammalian gamma-glutamate semialdehyde dehydrogenase activity is linear with respect to time and protein concentration. Comparison between the kinetic parameters reported for the bacterial enzyme and those reported here for the mammalian enzyme indicate similarities in the pH optima as well as a requirement for phosphate. Kinetic studies on mammalian enzyme yield apparent Km values of 1.8 mM for pyrroline 5-carboxylate, 0.2 mM for NADP+, and 11.3 mM for phosphate.     

Plasmodium falciparum: gene structure and hydropathy profile of the major merozoite surface antigen (gp195) of the Uganda-Palo Alto isolate

The gene encoding the 195,000-Da major merozoite surface antigen (gp195) of the FUP (Uganda-Palo Alto) isolate of Plasmodium falciparum, a strain widely used for monkey vaccination experiments, has been cloned and sequenced. The translated amino acid sequence of the FUP gp195 protein is closely related to the sequences of corresponding proteins of the CAMP (Malaysia) and MAD-20 (Papua New Guinea) isolates and more distantly related to those of the Wellcome (West Africa) and K1 (Thailand) isolates, supporting the proposed allelic dimorphism of gp195 within the parasite population. The prevalence of dimorphic sequences within the gp195 protein suggests that many gp195 epitopes would be group-specific. Despite the extensive differences in amino acid sequence between gp195 proteins of these two groups, the hydropathy profiles of proteins representative of both groups are very similar. The conservation of overall secondary structure shown by the hydropathy profile comparison indicates that gp195 proteins of the various P. falciparum isolates are functionally equivalent. This information on the primary structure of the FUP gp195 protein will enable us to evaluate the possible roles of conserved, group-specific and variable epitopes in immunity to the blood stage of the malaria parasite.     

Quantification and characterization of regulin, a Mr-230,000 highly elongated protein of rabbit reticulocytes

Procedures are described by which regulin in rabbit reticulocytes was quantified and isolated in relatively large amounts. In these cells the protein occurs at a ratio of about 1.1-1.6 regulin monomers/spectrin tetramer, corresponding to 80,000-100,000 molecules of Mr-230,000 regulin/cell. Erythrocytes contain less than 12% of the amount of regulin in reticulocytes and the protein has not been detected in non-erythroid cells. Regulin was found primarily in the cytosolic fraction of lysed reticulocytes. It appears to be unusually sensitive to proteolysis by Ca2+-activated thiol proteases. Isolation of Mr-230,000 undegraded regulin was accomplished by the use of protease inhibitors including N-ethylmaleimide. A striking characteristic of regulin is its tendency to aggregate in neutral solution of low ionic strength. Physical studies of the isolated protein indicate that it has a highly elongated form in solution. The protein has no known enzymatic activity but was shown previously to interact with and increase the enzymatic activity of a protein phosphatase. The properties of regulin suggest that it may have a structural function but it appears to be physically and immunologically distinct from known proteins. It is suggested that regulin may contribute to a gel matrix within the cytoplasm of reticulocytes.     

Enhancement of Butanol Formation by Clostridium acetobutylicum in the Presence of Decanol-Oleyl Alcohol Mixed Extractants

Extractive fermentation has been proposed to enhance the productivity of fermentations that are end product inhibited. Unfortunately, good extractants for butanol, such as decanol, are toxic to Clostridium acetobutylicum. The use of mixed extractants, namely, mixtures of toxic and nontoxic coextractants, was proposed to circumvent this toxicity. Decanol appeared to inhibit butanol formation by C. acetobutylicum when present in a mixed extractant that also contained oleyl alcohol. However, maintenance of the pH at 4.5 alleviated the inhibition of butanol production and the consumption of butyrate during solventogenesis. A mixed extractant that contained 20% decanol in oleyl alcohol enhanced butanol formation by 72% under pH-controlled conditions. The production of acetone and acetoin was also increased, even though these two products were not extractable. The enhancement of butanol formation was not limited by the toxicity of decanol. Supplementation of glucose and butyrate in the extractive fermentation yielded a 47% increase in butanol. The enhancement of butanol formation appeared to be dependent on the presence of dissolved decanol in the broth but was not observed unless an organic phase was present to extract butanol. A mechanism for the effects of decanol on product formation is proposed.     

Secondary production of an intertidal mussel (Mytilus edulis L.) population in the Eastern Scheldt (S.W. Netherlands)

We monitored an intertidal mussel (Mytilus edulis L.) population between June 1981 and June 1982 in the Eastern Scheldt estuary (S.W. Netherlands). Density and biomass of the population remained relatively constant over the study period. The shell length growth was described by a Gompertz growth curve. The parameters of this equation were estimated from a log-log-modified Ford-Walford plot of the growth-ring data. The slope of the relationship between animal weight and shell length is season-dependent, mainly due to the spawning cycle in larger mussels.Secondary production is estimated with the growth rate method. In the calculated growth rates the change in slope of the length-weight relationship is incorporated, as well as differences in length growth rates between summer and winter. Secondary production amounts to 156 g AFDW m^–2a ^–1 (expressed per m² of mussel bank). P:B is 0.50 a^–1. The mussel productivity is probably a limiting factor for the density of overwintering Oystercatchers (Haematopus ostralegus).     

Characterization and isolation of a trypsin-like serine protease from a long-term culture cytolytic T cell line and its expression by functionally distinct T cells

We describe the characterization and purification of a trypsin-like serine protease isolated from cloned long-term culture cytolytic T cell line (CTLL AK). High amounts of proteolytic activity were isolated from extracts of CTLL AK after either nitrogen cavitation or detergent lysis. Trypsin-like protease was detected by using either the ester compound N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester or a panel of low molecular amide substrates. The latter compounds were preferentially cleaved at the carboxyl termini of lysine and arginine residues. The enzyme activity was completely inhibited by two serine esterase inhibitors, diisopropylfluorophosphate and phenylmethanesulfonyl fluoride, and by aprotinin and meta-aminobenzamidine, which are known to block trypsin-like proteases. The pH optimum for CTLL AK-derived protease activity is 8 to 9. Analysis of the enzyme by gel filtration revealed that the cell-bound proteolytic activity was associated with a complex that could not be dissociated by treatment with Triton X-100. The CTLL AK-derived protease activity was found to reside in two proteins with relative molecular masses (Mr) of 32,000 and 40,000 daltons as determined by affinity labeling with [3H]diisopropylfluorophosphate and sodium dodecyl sulfate gel electrophoresis. High levels of enzyme activity were found in a panel of H-Y-specific cloned T cell lines with either cytolytic/suppressor (CTLL) or helper potential (THL), indicating a lack of correlation between trypsin-like protease activity and a particular T cell function. High enzyme activity was also detected in tumorigenic variants of CTLL. Furthermore, it was excluded that the trypsin-like activity detected was attributable to plasminogen activator activity. In contrast to cloned T effector cells and their in vitro or in vivo derived variants, considerably less activity was found in normal nonactivated or activated lymphocyte populations. The possible role of the trypsin-like serine protease in the function of T effector cells is discussed.     

Investigation of Dopamine Content, Synthesis, and Release in the Rabbit Retina In Vitro: I. Effects of Dopamine Precursors, Reserpine, Amphetamine, and l-DOPA Decarboxylase and Monoamine Oxidase Inhibitors

The basal catecholamine content of rabbit retina was determined by liquid chromatography with electrochemical detection (LC-EC) and 3,4-dihydroxyphenylethylamine (dopamine, DA) found to be the major catecholamine. The immediate DA precursor, 3,4-dihydroxyphenylalanine (L-DOPA), and the metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), were also detected at about 2.8% and 17% of DA levels, respectively. When added exogenously, L-tyrosine did not increase the rate of DA synthesis over the basal level. In contrast, exogenous L-DOPA led to a 3.5-fold increase in DA, and to a 20-fold increase in DOPAC content. The monoamine oxidase inhibitors pargyline and (-)-deprenyl differentially affected the degradation of DA, since 100 microM pargyline was apparently more effective than 100 microM (-)-deprenyl. Reserpine and (+/-)-amphetamine each induced a Ca2+-independent decrease of DA stores. The separate actions of reserpine and (+/-)-amphetamine in lowering tissue DA levels were additive, suggesting two separate pools of DA available for release from presynaptic stores. The present study demonstrates that the LC-EC technique may be used to investigate the modulation of the synthesis and release of retinal DA in vitro, without the prior uptake of radiolabelled transmitter.