A Highlights from MBoC Selection: A separable domain of the p150 subunit of human chromatin assembly factor-1 promotes protein and chromosome associations with nucleoli

Chromatin assembly factor-1 (CAF-1) is a three-subunit protein complex conserved throughout eukaryotes that deposits histones during DNA synthesis. Here we present a novel role for the human p150 subunit in regulating nucleolar macromolecular interactions. Acute depletion of p150 causes redistribution of multiple nucleolar proteins and reduces nucleolar association with several repetitive element–containing loci. Of note, a point mutation in a SUMO-interacting motif (SIM) within p150 abolishes nucleolar associations, whereas PCNA or HP1 interaction sites within p150 are not required for these interactions. In addition, acute depletion of SUMO-2 or the SUMO E2 ligase Ubc9 reduces α-satellite DNA association with nucleoli. The nucleolar functions of p150 are separable from its interactions with the other subunits of the CAF-1 complex because an N-terminal fragment of p150 (p150N) that cannot interact with other CAF-1 subunits is sufficient for maintaining nucleolar chromosome and protein associations. Therefore these data define novel functions for a separable domain of the p150 protein, regulating protein and DNA interactions at the nucleolus.     

Studies on the mechanism of activation of human natural killer function by interferon and inhibitors of thymidylate synthesis

Previous publications from this laboratory have demonstrated that agents such as methotrexate (MTX), 5-fluorodeoxyuridine (FUdR), trimethoprim, and D-glucosamine (D-GlcN), which are known to inhibit thymidylate synthesis, can augment human NK activity in vitro. Furthermore, this augmentation was inhibited by exogenous thymidine (TdR) at concentrations of 10(-6) to 10(-7) M. In this report, underlying mechanisms of action of FUdR, D-GlcN, and IFN are compared. Each of these agents increased the lytic activity of effector cells bound to targets but did not increase the percentage of conjugates formed. The augmentation could be induced in a population highly enriched for NK cells (Leu-1 lb positive in phenotype). FUdR and D-GlcN could not induce any augmentation in a Leu-1 lb-negative subpopulation whereas IFN could induce significant lytic activity. alpha-Amanitin, an inhibitor of RNA polymerase II, blocked the activation of NK activity by all three reagents; hence gene expression was required. Comparison of [35S]methionine-labeled proteins by two-dimensional gel electrophoresis revealed that six new proteins were induced in IFN-treated cells. Three of these were similar in pI and molecular weight to the newly synthesized proteins in the D-GlcN-treated cells. One protein was synthesized in increased amounts in the FuDR-treated cells and it was not common to either of the other treatments. Evidence to date is consistent with the hypothesis that separate mechanisms underlie the activation of NK cells by IFN and thymidylate synthesis inhibitors, although the existence of a final common pathway for all NK response modulators cannot be excluded at the present time.     

Evidence for Acquisition in Nature of a Chromosomal 2,4-Dichlorophenoxyacetic Acid/(alpha)-Ketoglutarate Dioxygenase Gene by Different Burkholderia spp

We characterized the gene required to initiate the degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) by the soil bacterium Burkholderia sp. strain TFD6, which hybridized to the tfdA gene of the canonical 2,4-D catabolic plasmid pJP4 under low-stringency conditions. Cleavage of the ether bond of 2,4-D by cell extracts of TFD6 proceeded by an (alpha)-ketoglutarate-dependent reaction, characteristic of TfdA (F. Fukumori and R. P. Hausinger, J. Bacteriol. 175:2083-2086, 1993). The TFD6 tfdA gene was identified in a recombinant plasmid which complemented a tfdA transposon mutant of TFD6 created by chromosomal insertion of Tn5. The plasmid also expressed TfdA activity in Escherichia coli DH5(alpha), as evidenced by enzyme assays with cell extracts. Sequence analysis of the tfdA gene and flanking regions from strain TFD6 showed 99.5% similarity to a tfdA gene cloned from the chromosome of a different Burkholderia species (strain RASC) isolated from a widely separated geographical area. This chromosomal gene has 77.2% sequence identity to tfdA from plasmid pJP4 (Y. Suwa, W. E. Holben, and L. J. Forney, abstr. Q-403, in Abstracts of the 94th General Meeting of the American Society for Microbiology 1994.). The tfdA homologs cloned from strains TFD6 and RASC are the first chromosomally encoded 2,4-D catabolic genes to be reported. The occurrence of highly similar tfdA genes in different bacterial species suggests that this chromosomal gene can be horizontally transferred.     

Use of spectral analysis to test hypotheses on the origin of pinnipeds

The evolutionary origin of the pinnipeds (seals, sea lions, and walruses) is still uncertain. Most authors support a hypothesis of a monophyletic origin of the pinnipeds from a caniform carnivore. A minority view suggests a diphyletic origin with true seals being related to the mustelids (otters and ferrets). The phylogenetic relationships of the walrus to other pinniped and carnivore families are also still particularly problematic. Here we examined the relative support for mono- and diphyletic hypotheses using DNA sequence data from the mitochondrial small subunit (12S) rRNA and cytochrome b genes. We first analyzed a small group of taxa representing the three pinniped families (Phocidae, Otariidae, and Odobenidae) and caniform carnivore families thought to be related to them. We inferred phylogenetic reconstructions from DNA sequence data using standard parsimony and neighbor-joining algorithms for phylogenetic inference as well as a new method called spectral analysis (Hendy and Penny) in which phylogenetic information is displayed independently of any selected tree. We identified and compensated for potential sources of error known to lead to selection of incorrect phylogenetic trees. These include sampling error, unequal evolutionary rates on lineages, unequal nucleotide composition among lineages, unequal rates of change at different sites, and inappropriate tree selection criteria. To correct for these errors, we performed additional transformations of the observed substitution patterns in the sequence data, applied more stringent structural constraints to the analyses, and included several additional taxa to help resolve long, unbranched lineages in the tree. We find that there is strong support for a monophyletic origin of the pinnipeds from within the caniform carnivores, close to the bear/raccoon/panda radiation. Evidence for a diphyletic origin was very weak and can be partially attributed to unequal nucleotide compositions among the taxa analyzed. Subsequently, there is slightly more evidence for grouping the walrus with the eared seals versus the true seals. A more conservative interpretation, however, is that the walrus is an early, but not the first, independent divergence from the common pinniped ancestor.      

A novel means to develop strain-specific DNA probes for detecting bacteria in the environment.

A simple means to develop strain-specific DNA probes for use in monitoring the movement and survival of bacteria in natural and laboratory ecosystems was developed. The method employed amplification of genomic DNA via repetitive sequence-based PCR (rep-PCR) using primers specific for repetitive extragenic palindromic (REP) elements, followed by cloning of the amplified fragments. The cloned fragments were screened to identify those which were strain specific, and these were used as probes for total genomic DNA isolated from microbial communities and subjected to rep-PCR. To evaluate the utility of the approach, we developed probes specific for Burkholderia cepacia G4 and used them to determine the persistence of the strain in aquifer sediment microcosms following bioaugmentation. Two of four probes tested were found to specifically hybridize to DNA fragments of the expected sizes in the rep-PCR fingerprint of B. cepacia G4 but not to 64 genetically distinct bacteria previously isolated from the aquifer. One of these probes, a 650-bp fragment, produced a hybridization signal when as few as 10 CFU of B. cepacia G4 were present in a mixture with 10(6) CFU nontarget strains, indicating that the sensitivity of these probes was comparable to those of other PCR-based detection methods. The probes were used to discriminate groundwater and microcosm samples that contained B. cepacia G4 from those which did not. False-positive results were obtained with a few samples, but these were readily identified by using hybridization to the second probe as a confirmation step. The general applicability of the method was demonstrated by constructing probes specific to three other environmental isolates.     

Responses and locations of neurones in the locust metathoracic femoral chordotonal organ

Summary Insect legs possess chordotonal organs which monitor leg angle, and the direction, velocity and acceleration of leg movements. The locust metathoracic femoral chordotonal organ (mtFCO) has previously been studied morphologically and physiologically, but no detailed analysis of the responses of individual neurones, and their location in the organ has so far been produced. By recording from, and staining mtFCO neurones I have been able to compile for the first time such a map. The distribution of neurone somata in the locust mtFCO is more complex than previously thought: receptors sensitive to both stretch and relaxation of the apodeme are distributed throughout the organ. Seventeen response types were encountered. Neurones with a particular response type have somata in comparable locations within the mtFCO. Comparisons are made between the response types found in the stick insect and those in the locust. The possible functions of some of the responses are discussed.Abbreviation (mt)FCO (metathoracic) femoral chordotonal organ - F-T femur-tibia