Crustacean plankton in Høylandet

Crustacean plankton was studied in 12 lakes in theHøylandet area in 1986–87. Basic lake characteristicsare elevation 134–415 m, surface area 9–530 ha, pH 5.9–7.1,conductivity (25 °C) 12–40 µS cm^-1 andSecchi depth 4–9 m. Number ofspecies present varied between 3 and 11. Populationnumbers between 4000 and 400 000 per m² andbiomasses were within the range 30 to1800 mg m^-2 dry weight. Cladocera dominated overCopepoda in lakes with allopatric brown trout (Salmo trutta L.), on the contrary to lakes also populatedby Arctic charr (Salvelinus alpinus (L.)). Thesevariations are caused by differences in elevation,lake morphometry, water quality, fish predation andthe general distribution of the species. The largestlakes at lowest elevation were richest in species. Theacid sensitive genus Daphnia was represented by 3species. The lakes Storgrønningen (530 ha) andRøyrtjønna (27 ha) were sampled monthly in theice-free seasons of 1986–89, and Storgrønningen moreintensively from June to November in 1987 and 1988. The same6 species of Cladocera and 5 of Copepoda were presentin both lakes. Their life cycles were traditional orknown from several other Scandinavian lakes. Meanseasonal biomasses were of the range600–750 mg m^-2. At the species level, there wereconsiderable variations between years inStorgrønningen and particularly in Røyrtjønna. Noeffects of human impacts on the crustacean planktonwere found. The Høylandet lakes are representative forScandinavian oligotrophic to almost ultra-oligotrophiclakes. Storgrønningen is well qualified as a referencesystem. The between year variations in Røyrtjønna areso extreme, that any human impact could only be traced at alevel causing the extinction of species.     

DNA-Protein Complex in Circular DNA from Phage ϕ29

THE DNA of the B. subtilis phage ?29 has been described as unpermuted linear duplex molecules¹ of molecular weight 11 × 10⁶, but the formation of circular molecules has also been indicated, suggesting the existence of cohesive ends^1,2.     

Synthesis of Bacteriophage φ29 Proteins in Bacillus subtilis

Seventeen bacteriophage phi29 proteins were detected in ultraviolet light-irradiated Bacillus subtilis by autoradiography of polyacrylamide slab gels. The appearance of phi29 proteins occurred either before or concomitantly with viral DNA replication. Viral proteins detected early in the infectious cycle consisted of nine polypeptides ranging from 5,200 daltons to 54,000 daltons. Two of the early proteins were identified as, respectively, the major capsid protein and the protein comprising the filaments which extend from the head of the virus. Late phi29 proteins were composed of eight polypeptides ranging from 14,000 daltons to 95,000 daltons. Only three late proteins were noncapsid proteins. Among the early proteins, six were synthesized at diminishing rates late in the infectious cycle. One of the early proteins (protein 12) lacked histidine, whereas two (proteins 10 and 15) lacked tryptophan. Among the 17 proteins detected, 10 were viral noncapsid proteins. The amount of viral genetic information required to code for the 17 proteins detected in these experiments (81% of the potential genetic information of phi29 DNA) compares favorably with the genetic information detected as mRNA in a previous report, 85% of the potential information on the phi29 chromosome.     

Molecular dissection of ø29 scaffolding protein function in an in vitro assembly system

An in vitro assembly system was developed to study prolate capsid assembly of phage ?29 biochemically, and to identify regions of scaffolding protein required for its functions. The crowding agent polyethylene glycol can induce bacteriophage ?29 monomeric capsid protein and dimeric scaffolding protein to co-assemble to form particles which have the same geometry as either prolate T=3 Q=5 procapsids formed in vivo or previously observed isometric particles. The formation of particles is a scaffolding-dependent reaction. The balance between the fidelity and efficiency of assembly is controlled by the concentration of crowding agent and temperature. The assembly process is salt sensitive, suggesting that the interactions between the scaffolding and coat proteins are electrostatic. Three N-terminal ?29 scaffolding protein deletion mutants, Delta 1-9, Delta 1-15 and Delta 1-22, abolish the assembly activity. Circular dichroism spectra indicate that these N-terminal deletions are accompanied by a loss of helicity. The inability of these proteins to dimerize suggests that the N-terminal region of the scaffolding protein contributes to the dimer interface and maintains the structural integrity of the dimeric protein. Two C-terminal scaffolding protein deletion mutants, Delta 79-97 and Delta 62-97, also fail to promote assembly. However, the secondary structure and the dimerization ability of these mutants are unchanged relative to wild-type, which suggests that the C terminus is the likely site of interaction with the capsid protein.     

H1° and H3.3B mRNA levels in developing rat brain

Two overlapping rat cDNAs, covering a continuous region of 1107 base pairs, have been isolated and sequenced. The clones contain identical open reading frames, encoding a 136 amino acid long polypeptide which exhibits 100% identity to other mammalian H3.3 histone variants. We show that the inserts derive, in particular, from the H3.3B gene. We used these inserts and an insert from an H1° encoding clone, previously described (6), as probes to study the accumulation of mRNAs encoding the corresponding histone replacement variants (namely, H1° and H3.3) during rat brain development. We found that the concentration of both H1° and H3.3B mRNAs decreases from the embryonal day 18 (E18) to the postnatal day 10 (P10), with inverse correlation to protein accumulation.This paper is dedicated to our friend Paolo Carbone who devoted his life to research and teaching in Genetics. We will always remember him for scientific honesty and for his unique qualities of humanity.     

Role of φ29 connector channel loops in late-stage DNA packaging

Double-stranded DNA bacteriophages and their eukaryotic virus counterparts have 12-fold head-tail connector assemblages embedded at a unique capsid vertex. This vertex is the site of assembly of the DNA packaging motor, and the connector has a central channel through which viral DNA passes during genome packaging and subsequent host infection. Crystal structures of connectors from different phages reveal either disordered residues or structured loops that project into the connector channel. Given the proximity to the translocating DNA substrate, these loops have been proposed to play a role in DNA packaging. Previous models have proposed structural motions in either the packaging ATPase or the connector channel loops as the driving force that translocates the DNA into the prohead. Here, we mutate the channel loops of the Bacillus subtilis bacteriophage φ29 connector and show that these loops have no active role in translocation of DNA. Instead, they appear to have an essential function near the end of packaging, acting to retain the packaged DNA in the head in preparation for motor detachment and subsequent tail assembly and virion completion.     

Temperature-Shift Analysis of Bacteriophage φ29 Gene Expression in Bacillus amyloliquefaciens

Temperature shift-up experiments with conditional lethal mutants of Bacillus phage phi29 have allowed placement of early, middle, and late functions on the linear phi29 genetic map Most of the phi29 cistrons are late and are found at the ends of the map.     

Quantitative and time-course analysis of microbial degradation of 1H,1H,2H,2H,8H,8H–perfluorododecanol in activated sludge

A methylene group in the fluorinated carbon backbone of 1H,1H,2H,2H,8H,8H–perfluorododecanol (degradable telomer fluoroalcohol, DTFA) renders the molecule cleavable by microbial degradation into two fluorinated carboxylic acids. Several biodegradation products of DTFA are known, but their rates of conversion and fates in the environment have not been determined. We used liquid chromatography coupled with tandem mass spectrometry (LC/MS/MS) to quantitatively investigate DTFA biodegradation by the microbial community in activated sludge in polyethylene terephthalate (PET) flasks, which we also determined here showed least adsorption of DTFA. A reduction in DTFA concentration in the medium was accompanied by rapid increases in the concentrations of 2H,2H,8H,8H–perfluorododecanoic acid (2H,2H,8H,8H–PFDoA), 2H,8H,8H-2-perfluorododecenoic acid (2H,8H,8H-2-PFUDoA), and 2H,2H,8H-7-perfluorododecenoic acid and 2H,2H,8H-8-perfluorododecenoic acid (2H,2H,8H-7-PFUDoA/2H,2H,8H-8-PFUDoA), which were in turn followed by an increase in 6H,6H–perfluorodecanoic acid (6H,6H–PFDeA) concentration, and decreases in 2H,2H,8H,8H–PFDoA, 2H,8H,8H-2-PFUDoA, and 2H,2H,8H-7-PFUDoA/2H,2H,8H-8-PFUDoA concentrations. Accumulation of perfluorobutanoic acid (PFBA), a presumed end product of DTFA degradation, was also detected. Our quantitative and time-course study of the concentrations of these compounds reveals main routes of DTFA biodegradation, and the presence of new biodegradation pathways.

     

Determination of Hα,Hβ and Hβ,C′ coupling constants in13C-labeled proteins

Summary A sensitive method to assign H protons stereospecifically as well as to determine rotamer populations about ₁, in two 3D experiments is presented. The SOFT-HCCH-COSY experiment allowed us to measure the³J(H,C) couplings, using constant time evolution of C in t₂ and C_aliphatic-selective decoupling during t₃. The SOFT-HCCH-E.COSY experiment allowed us to measure the³J(H,H) couplings, using constant time evolution of C in t₂, a small flip angle¹H excitation pulse in the second mixing time, and double-band-selective decoupling (aliphatic and carbonyl carbons) during t₃. The method was applied to ribonuclease T₁.     

Critical Function of γH2A in S-Phase

Phosphorylation of histone H2AX by ATM and ATR establishes a chromatin recruitment platform for DNA damage response proteins. Phospho-H2AX (γH2AX) has been most intensively studied in the context of DNA double-strand breaks caused by exogenous clastogens, but recent studies suggest that DNA replication stress also triggers formation of γH2A (ortholog of γH2AX) in Schizosaccharomyces pombe. Here, a focused genetic screen in fission yeast reveals that γH2A is critical when there are defects in Replication Factor C (RFC), which loads proliferating cell nuclear antigen (PCNA) clamp onto duplex DNA. Surprisingly Chk1, Cds1/Chk2 and the Rad9-Hus1-Rad1 checkpoint clamp, which are crucial for surviving many genotoxins, are fully dispensable in RFC-defective cells. Immunoblot analysis confirms that Rad9-Hus1-Rad1 is not required for formation of γH2A by Rad3/ATR in S-phase. Defects in DNA polymerase epsilon, which binds PCNA in the replisome, also create an acute need for γH2A. These requirements for γH2A were traced to its role in docking with Brc1, which is a 6-BRCT-domain protein that is structurally related to budding yeast Rtt107 and mammalian PTIP. Brc1, which localizes at stalled replication forks by binding γH2A, prevents aberrant formation of Replication Protein A (RPA) foci in RFC-impaired cells, suggesting that Brc1-coated chromatin stabilizes replisomes when PCNA or DNA polymerase availability limits DNA synthesis.