Two-dimensional display of lambda and Escherichia coli restriction fragments separated by Hg-Cs2SO4 centrifugation and gel electrophoresis

EcoRI restriction fragments derived from the DNA of bacteriophage lambda and Escherichia coli were fractionated by density gradient centrifugation of their mercury complexes in Cs2SO4 and subsequent electrophoresis on a horizontal agarose-gel slab. In this two-dimensional display, lambda fragments were resolved into six components and E coli fragments into more than 108 components. Bacterial chromosome regions contiguous to lambda prophage integrated at different sites were amplified by induction, and the EcoRI fragments were subjected to the two-dimensional analysis. As expected, the sets of amplified fragments were clearly different among the various lysogens. The approximate genome region affected by induction was estimated as one-tenth of the whole chromosome.     

Endo16 is required for gastrulation in the sea urchin Lytechinus variegatus

The Endo16 gene encodes a large extracellular protein with several functional domains that provide some insight into the role of this protein during embryonic development. We isolated the full-length cDNA sequence from Lytechinus variegatus and utilized morpholinos to further investigate the role of Endo16 during embryonic development in this species. Endo16-deficient embryos failed to undergo gastrulation and the blastocoele became filled with dissociated cells after 24 h of incubation. Moreover, there was a delay in endoderm differentiation as assayed by staining with an antibody that recognizes Endo1. The differentiation of other cell types including oral ectoderm, primary mesenchymal cells (PMC) and secondary mesenchymal cells (SMC) appeared to be normal, although the patterns of protein expression did not resemble control embryos due to the gross morphological abnormalities elicited by the LvEndo16 morpholino. Microinjection of full-length EGFP mRNA with the LvEndo16 morpholino-targeted sequence confirmed that this phenotype can be attributed specifically to the loss of Endo16 protein. Taken together, our data suggest that Endo16 may be required for the cell-extracellular matrix (ECM) interactions that are required for endoderm differentiation in the sea urchin embryo.     

Select light spectra affect gonad color in the sea urchin Lytechinus variegatus

It is suggested that gonad color in sea urchins depends upon the in vivo accumulation and metabolism of red and yellow carotenoid pigments. We hypothesized that differential light exposure could affect carotenoid deposition and, hence, gonad color in sea urchins. We therefore performed two experiments to determine whether light spectra affect the gonad color of Lytechinus variegatus. In the first experiment, urchins were fed a formulated feed supplemented with or without β-carotene and held beneath three lighting regimes designed to emit differing wavelengths of the visible spectrum. After 12 weeks, urchins were dissected and gonad color (CIE L*a*b*) was measured with a Pantone Capsure RM200. Actinic light significantly increased the value of a* (red) in gonad color. Color in the orange and yellow spectra in the gonads increased in individuals fed the β-carotene supplemented diet. In the second experiment, we cultured urchins for nine weeks under lamps specialized to emit UV radiation. All urchins in this experiment received diets supplemented with β-carotene. There was no significant difference in harvested gonad color between these treatments. These data suggest that light quality and dietary carotenoids affect carotenoid deposition in the gonads.     

Effect of ultraviolet radiation on growth and percent settlement of larval Lytechinus variegatus (Echinodermata: Echinoidea)

Lytechinus variegatus larvae were used to examine the effects of ecologically relevant short-term exposures of ultraviolet radiation (UVR) on larval morphology and settlement. A laboratory experiment tested the blastula, gastrula, and early pluteus stages of larval development for susceptibility to damage from exposure to UVR produced by an artificial light source. Larval post-oral arm length and percent settlement were measured to assess UVR effects. Larvae exposed at the blastula and gastrula stages had a reduction in larval post-oral arm length compared with non-exposure controls, while larvae exposed at the early pluteus stage were similar to controls. Larvae exposed in the gastrula stage had a significant reduction in percent settlement compared with controls. Negative consequences from ecologically relevant levels of UVR were differentially dependent on the larval stage at time of exposure suggesting time point in larval development at exposure will be important in interpreting effects on higher levels of biological organization.     

Bioelectric responses at fertilization: Separation of the events associated with insemination from those due to the cortical reaction in sea urchin,Lytechinus variegatus

The bioelectric responses at fertilization of the sea urchin Lytechinus variegatus are a complex series of membrane potential and resistance changes that occur concomitant with gamete fusion, ionic fluxes, and the cortical granule discharge. This work attempts to separate the electrical effects of sperm-egg interactions from those of the cortical reactions. Two approaches were taken to discern the electrical events associated with insemination, distinct from cortical granule discharge: (1) fertilization of eggs treated with 3% urethane, 10 mM procaine, or 10 mM nicotine, to prevent the cortical reaction and (2) refertilization of fertilized eggs (denuded with 1 mM aminotriazole containing 1 mg/ml soybean trypsin inhibitor). Cortical granule discharge in the absence of sperm incorporation was investigated by artificial activation with 5 μM A23187 or by fertilization in the presence of 10 μM cytochalasin D, which prevents incorporation. These results are consistent with a model in which the sperm-egg interaction triggers both a rapid (50–400 msec), but minor (?10 mV), electrical transient that leads to an action potential and then both the Na⁺-dependent fast block to polyspermy and the late block resulting from the secretion of the cortical granules.     

Restriction mapping of the DNA of the Streptomyces temperate phage phi C31 and its derivatives

DNA of phi C31 propagated on Streptomyces lividans 66 contained no sites for the restriction enzymes BamHI, SalPI (=PstI) and XhoI; one for XbaI; three for HpaI; five for ClaI and KpnI; six for EcoRI; about 13 for HindIII; about 14 for BclI; and more than 15 for FspAI, HgiAI, SacI, SalGI and SmaI. A complete map of 20 sites (XbaI, HapI, ClaI, KpnI and EcoRI) was obtained using partial digestion and double digestion of DNA of the wild-type and deletion and insertion mutants. The total molecular size was estimated to be 41.2 kb.     

Effects of hypercapnia on aspects of feeding,nutrition, and growth in the edible sea urchin Lytechinus variegatus held in culture

Land-based aquaculture facilities experience occasional hypercapnic conditions due to the accumulation of the metabolic waste product carbon dioxide. Pre-gonadal Lytechinus variegatus (horizontal diameter?=?20?mm) were exposed to control (608?μatm pCO₂, pH 8.1) or hypercapnic conditions (1738?μatm pCO₂, pH 7.7) in synthetic seawater for 14?weeks. Sea urchins exposed to hypercapnic conditions exhibited significantly slower growth (reduced dry matter production), primarily due to reduced test production. Higher fecal production rates and lower ash absorption efficiency (%) in individuals exposed to hypercapnic conditions suggest the ability to process or retain dietary carbonates may have been affected. Significant increases in neutral lipid storage in the gut and increased soluble protein storage in the gonads of individuals exposed to hypercapnic conditions suggest alterations in nutrient metabolism and storage. Furthermore, organic production and energy allocation increased in the lantern of those individuals exposed to hypercapnic conditions. These results suggest chronic exposure to hypercapnic conditions alters nutrient allocation to organ systems and functions, leading to changes in somatic and reproductive production.     

Cloning and characterization of a phospholipase C-beta isoform from the sea urchin Lytechinus pictus

Calcium is a ubiquitous intracellular signaling molecule controlling a wide array of cellular processes including fertilization and egg activation. The mechanism for triggering intracellular Ca(2+) release in sea urchin eggs during fertilization is the generation of inositol-1,4,5-trisphosphate by phospholipase C (PLC) hydrolysis of phosphatidylinositol-4,5-bisphosphate. Of the five PLC isoforms identified in mammals (beta, gamma, delta, epsilon and zeta), only PLCgamma and PLCdelta have been detected in echinoderms. Here, we provide direct evidence of the presence of a PLCbeta isoform, named suPLCbeta, within sea urchin eggs. The coding sequence was cloned from eggs of Lytechinus pictus and determined to have the greatest degree of homology and identity with the mammalian PLCbeta4. The presence of suPLCbeta within the egg was verified using a specifically generated antibody. The majority of the enzyme is localized in the non-soluble fraction, presumably the plasma membrane of the unfertilized egg. This distribution remains unchanged 1 min postfertilization. Unlike PLCbeta4, suPLCbeta is activated by G protein betagamma subunits, and this activity is Ca(2+)-dependent. In contrast to all known PLCbeta enzymes, suPLCbeta is not activated by Galphaq-GTPgammaS subunit suggesting other protein regulators may be present in sea urchin eggs.     

A developmentally regulated α2,8-polysialyltransferase in embryos of the sea urchin Lytechinus pictus

The recent chemical identification of polysialylated glycoproteins in the jelly coat and on the cell surface of the sea urchin egg raises important questions about their biosynthesis and possible function. Using CMP-[¹⁴C]-Neu5Ac as substrate and cell free preparations from eggs and embryos of the sea urchin Lytechinus pictus , we have identified a membrane associated CMP-Neu5Ac:poly-α2,8 sialosyl sialyltransferase (polyST) that transferred Neu5Ac from CMP-Neu5Ac to an endogenous acceptor membrane protein of approximately 38kDa. An average of five to six [¹⁴C]-Neu5Ac residues were transferred to the glycan moiety of this protein. The membrane-associated polyST also catalyzed the polysialylation of several exogenous mammalian ganglioside acceptors, including G_D3. Given that no structurally similar naturally occurring polysialylated gangliosides have been described, nor were observed in the present study, we conclude that a single polyST activity catalyzes sialylation of the endogenous acceptor protein and the gangliosides. Using an excess of G_D3 as an exogenous acceptor, it was established that the expression of the polyST in L. pictus embryos increased rapidly at the mesenchyme blastula stage and reached a maximum at the gastrula stage. The finding that this polyST in the sea urchin embryo is developmentally regulated raises the possibility that it may play a role in the changing cell and tissue interactions that occur during gastrulation and the early stages of spicule formation.     

Organization of the ribosomal RNA gene cluster in Aspergillus nidulans

DNA coding for ribosomal RNA in Aspergillus nidulans was found to consist of a unit 7.8 kb in size which is tandemly repeated in the genome and codes for 5.8S, 18S and 26S rRNA. The repeat unit has been cloned, and its restriction map and the location of the individual rRNA coding sequences within the unit have been established.     

Periodicities in coding and noncoding regions of the genes

Gene population statistical studies of protein coding genes and introns have identified two types of periodicities on the purine/pyrimidine alphabet: (i) the modulo 3 periodicity or coding periodicity (periodicity P3) in protein coding genes of eukaryotes, prokaryotes, viruses, chloroplasts, mitochondria, plasmids and in introns of viruses and mitochondria, and (ii) the modulo 2 periodicity (periodicity P2) in the eukaryotic introns. The periodicity study is herein extended to the 5' and 3' regions of eukaryotes, prokaryotes and viruses and shows: (i) the periodicity P3 in the 5' and 3' regions of eukaryotes. Therefore, these observations suggest a unitary and dynamic concept for the genes as for a given genome, the 5' and 3' regions have the genetic information for protein coding genes and for introns: (1) In the eukaryotic genome, the 5' (P2 and P3) and 3' (P2 and P3) regions have the information for protein coding genes (P3) and for introns (P2). The intensity of P3 is high in 5' regions and weak in 3' regions, while the intensity of P2 is weak in 5' regions and high in 3' regions. (2) In the prokaryotic genome, the 5' (P3) and 3' (P3) regions have the information for protein coding genes (P3). (3) In the viral genome, the 5' (P3) and 3' (P3) regions have the information for protein coding genes (P3) and for introns (P3). The absence of P2 in viral introns (in opposition to eukaryotic introns) may be related to the absence of P2 in 5' and 3' regions of viruses.     

Demonstration of the granular layer and the fate of the hyaline layer during the development of a sea urchin (Lytechinus variegatus)

Summary Employing electron-microscopic methods that help retain polyanionic materials, we describe the extracellular coverings of a sea urchin (Lytechinus variegatus) throughout ontogeny. The surface of the embryo is covered by a two-layered cuticle (commonly called the hyaline layer), which in turn is covered by a granular layer. The granular layer is retained after addition of alcian blue to the fixative solutions, and has not been previously described for any sea urchin. After hatching, the granular layer disappears, but the hyaline layer continues to cover most of the larval surface until settlement and metamorphosis. A few days before metamorphosis, the hyaline layer lining the vestibular invagination of the competent pluteus larva is replaced by a three-layered cuticle resembling that of the adult sea urchin. The hyaline layer covering the rest of the larva is evidently lost at metamorphosis during the involution of the general epidermis.     

Sequence of a yeast DNA fragment containing a chromosomal replicator and the TRP1 gene

The DNA sequence of a 1.45 kb EcoRI fragment from the yeast (Saccharomyces cerevisiae) TRP1 region has been determined. The fragment contains the TRP1 gene and a yeast chromosomal replicator. The TRP1 gene has been located on the fragment by analysis of potential initiation and termination codons in the DNA sequence. This location has been confirmed by subcloning portions of the fragment. Both the 5' and 3' noncoding regions of the TRP1 gene contain sequence homologies with analogous areas surrounding other yeast genes. The yeast replicator has been localized in a region near the 3' end of the TRP1 gene. The DNA sequence in this region contains several structural features which may be involved in the initiation of DNA replication.     

Polymorphisms in the coding and noncoding regions of murinePgk-1 alleles

The mouse X-linkedPgk-1 gene encodes phosphoglycerate kinase. When transfected into human cells, thePgk-1^b allele causes the appearance of mouse PGK-1^b enzyme activity. We describe here cloning of mousePgk-1^a, an allele ofPgk-1 which encodes an enzyme, PGK-1^a, with distinct electrophoretic mobility. We constructed recombinants between the DNA encodingPgk-1^b andPgk-1^a and transfected these constructs into human to assess the electrophoretic characteristics of each recombinant. In this way the charge variation between the two proteins was localized to exons 4 or 5. Sequencing of these exons revealed a single base-pair difference between the two alleles at codon 155, which predicts the amino acids lysine and threonine in PGK-1^b and PGK-1^a, respectively. A number of other DNA sequence polymorphisms exist betweenPgk-1^b andPgk-1^a including part of an L1 repeated element unique toPgk-1^a. This work was supported by the Medical Research Council of Canada, the National Cancer Institute of Canada, and the Deutsche Forschungsgemeinschaft, SFB 304.     

Effects of phenobarbital on protein synthesis and polysome levels in rat liver.

Administration of phenobarbital to rats increases the rate of synthesis of certain microsomal drug-metabolizing enzymes in a selective manner and promotes proliferation of smooth endoplasmic reticulum in the liver. Phenobarbital increased a number of factors by which protein synthesis could be enhanced in the liver. It produced a 30% increase in the amount of ribosomes and mRNA per cell. The proportion of ribosomes associated with polysomes was increased by 5-10% over normal liver. There was a 10-30% increase in the rate of ploypeptide elongation and a small increase or no change in polysome size, indicating that the rate of polypeptide initiation was increased proportionately. The product of these effects accounts for the 1.5-fold increase in the rate of total protein synthesis previously reported. The average polysome size, and the size of free polysomes in particular, was maintained when actinomycin D was administered to phenobarbital-pretreated rats, suggesting that the rate of mRNA degradation was decreased selectively. Phenobarbital did not, however, affect the distribution of ribosomes between the free and membrane-bound states or the activity of ribonucleases associated with isolated free and bound polysomes. Thus, we conclude that phenobarbital stimulates protein synthesis by expanding the mRNA pool, at least partially through effects on mRNA degradation, and by augmenting the rate of mRNA translation.     

Isolation and characterization of the dut gene of Escherichia coli. II. Restriction enzyme mapping and analysis of polypeptide products

Restriction endonuclease mapping of previously constructed dut plasmids has been carried out using the enzymes PvuI, PvuII and SacI. Various dut plasmids were also tested in the "maxicell" protein-synthesizing system. They all show two protein bands in common, one of Mr 16000 in agreement with the size previously reported for the purified dUTPase subunit (Shlomai and Kornberg, 1978). With the information obtained the structural gene for dUTPase can be assigned to a 950-bp SacI-PvuII fragment of the E. coli genome. Studies, described in the preceding paper, on the overproduction of dUTPase by bacterial strains carrying different dut plasmids strongly suggest that the dut gene is transcribed in the direction from the SacI site towards the PvuII site and that the SacI site is located within the dut control region. The second protein band observed in the "maxicell" experiments has an Mr of 23500. Its identity is unknown but it may represent a precursor of dUTPase or the product of a separate gene located between dut and pyrE.     

Molecular cloning and characterization of double-stranded cDNA coding for bovine chymosin

A full-length cDNA copy of the mRNA encoding calf chymosin (also known as rennin), a proteolytic enzyme with commercial importance in the manufacture of cheese, has been cloned in an f1 bacteriophage vector. The nucleotide sequence of the cDNA was determined, and translation of that sequence into amino acids predicts that the zymogen prochymosin is actually synthesized in vivo as preprochymosin with a 16 amino acid signal peptide. In vitro translation of total poly(A)-enriched RNA from the calf fourth stomach (abomasum) and immunoprecipitation with antichymosin antiserum revealed that a form of chymosin (probably preprochymosin judging from the M_r-value) is the major in vitro translation product of RNA from that tissue. Gel-transfer hybridization of restriction endonuclease-cleaved bovine chromosomal DNA with labeled cDNA probes indicated that the two known forms of chymosin, A and B, must be products of two different alleles of a single chymosin gene.     

The plasmid cloning vector pBR325 contains a 482 base-pair-long inverted duplication

The plasmid pBR325 is a cloning vector constructed in vitro by addition of the chloramphenicol resistance (Cmr) gene of an IS1-flanked transposon to pBR322 (Bolivar, 1978). It is a 5 995 bp plasmid carrying no sequence originating from IS1. DNA-sequence data suggest that its Cmr segment was derived from a Cm transposon longer than Tn9. The plasmid pBR325 carries between the Cmr and Tcr genes a 482 bp sequence which duplicates, in the opposite orientation, a section pf pBR322 located at the end of the tcr gene. The same structure was found in pBR328, a deletion derivative of pBR325 (Soberon et al., 1980). The possible implications of this inverted duplication on cloning experiments are discussed.