Chromatin structure regulates gene conversion

Homology-directed repair is a powerful mechanism for maintaining and altering genomic structure. We asked how chromatin structure contributes to the use of homologous sequences as donors for repair using the chicken B cell line DT40 as a model. In DT40, immunoglobulin genes undergo regulated sequence diversification by gene conversion templated by pseudogene donors. We found that the immunoglobulin Vλ pseudogene array is characterized by histone modifications associated with active chromatin. We directly demonstrated the importance of chromatin structure for gene conversion, using a regulatable experimental system in which the heterochromatin protein HP1 (Drosophila melanogaster Su[var]205), expressed as a fusion to Escherichia coli lactose repressor, is tethered to polymerized lactose operators integrated within the pseudo-Vλ donor array. Tethered HP1 diminished histone acetylation within the pseudo-Vλ array, and altered the outcome of Vλ diversification, so that nontemplated mutations rather than templated mutations predominated. Thus, chromatin structure regulates homology-directed repair. These results suggest that histone modifications may contribute to maintaining genomic stability by preventing recombination between repetitive sequences.     

EFFECT OF SALINITY ON PSEUDO‐NITZSCHIA SPECIES (BACILLARIOPHYCEAE) GROWTH AND DISTRIBUTION1

Salinity varies widely in coastal areas that often have a high abundance of Pseudo‐nitzschia H. Peragallo. Pseudo‐nitzschia is abundant in Louisiana waters, and high cellular domoic acid has been observed in natural samples but no human illness has been reported. To assess the threat of amnesic shellfish poisoning (ASP), we examined the effect of salinity on Pseudo‐nitzschia occurrence in the field and growth in the laboratory with special emphasis on the salinity range where oysters are harvested (10–20 psu). In Louisiana coastal waters, Pseudo‐nitzschia spp. occurred over a salinity range of 1 to >35 psu, but they occurred more frequently at higher rather than lower salinities. Seven species were identified, including toxigenic species occurring at low salinities. In culture studies, seven clones of three species grew over a salinity range of 15 to 40 psu, some grew at salinities down to 6.25 psu, and most grew at salinities up to 45 psu. Tolerance of low salinities decreased from Pseudo‐nitzschia delicatissima (Cleve) Heiden to P. multiseries (Hasle) Hasle to P. pseudodelicatissima (Hasle) Hasle emend. Lundholm, Hasle et Moestrup. In conclusion, although Pseudo‐nitzschia was more prevalent in the field and grew better in the laboratory at higher salinities, it grew and has been observed at low salinities. Therefore, the probability of ASP from consumption of oysters harvested from the low salinity estuaries of the northern Gulf of Mexico is low but not zero; animal mortality events from toxin vectors other than oysters at higher salinity on the shelf are more likely.     

The Role of <Emphasis Type="Italic">Habitus</Emphasis> in the Maintenance of Traditional Noongar Plant Knowledge in Southwest Western Australia

We examine the role that habitus, an individual’s or group’s dispositions, has played in the retention of traditional ecological knowledge among the Noongar people of south-western Australia. We sought to determine if current plant knowledge reflects Noongar habitus or, alternatively, the use of fall-back species that were important due to the intermittency of agricultural employment and the social exclusion of Aboriginal people up until at least the 1960s in Western Australia. We compared the seasonal availability of Noongar food plant resources currently known by Noongar Elders to those described at the time of European settlement. We used non-metric Multidimensional Scaling (nMDS) and multivariate statistics to compare the seasonal availability of plant resources with the seasonal availability of work prior to the introduction of civil rights for Aboriginal Australians in the 1960s. We show that the seasonal pattern of plant knowledge has changed little since settlement and that there was no significant relationship between the seasonal availability of work and plant knowledge. This result suggests that prior to 1960 Noongars maintained a reasonably traditional round of seasonal activities involving traditional plant use. We suggest that Noongar habitus guided their response to the colonising culture and helped preserve traditional ecological knowledge.     

Cytokine (tumor necrosis factor, IL-6, and IL-8) production by respiratory syncytial virus-infected human alveolar macrophages.

Human alveolar macrophages (AM) are susceptible to infection with respiratory syncytial virus (RSV), but the infection is abortive after the initial cycles of virus replication. We have investigated if RSV infection of AM results in the production of cytokines TNF, IL-6, and IL-8, all of which may modulate inflammatory and immune responses to the virus, as well as may directly protect respiratory epithelial cells against spread of infection. Within 1 h after interaction with RSV, increased mRNA levels were found for all three cytokines. Peak expression of the mRNAs occurred at 3 to 6 h. The virus most effectively induced TNF mRNA expression greater than IL-6 mRNA greater than IL-8 mRNA, as compared to cytokine mRNA expression induced by bacterial endotoxin. Inactivated virus was almost as effective as live virus in inducing and maintaining increased IL-6 and IL-8 mRNA over 16 h, whereas live infectious RSV was necessary for maintaining TNF mRNA expression over the same time. Protein concentrations of the different cytokines in the supernatants of infected AM reflected the increased levels of mRNA in the cells. Despite the high levels of cytokines with possible antiviral activity (TNF and IL-6) in the AM supernatants, neither supernatants nor rTNF when added to bronchial epithelial cells protected them from infection with RSV. However, TNF, IL-1, and RSV, but not IL-6, induced IL-8 and IL-6 mRNA expression by the bronchial epithelial cells suggesting that cytokines produced by RSV-infected AM may be more important in modulating the inflammatory response in infection than directly interfering with virus infection/replication of airway epithelium.     

A rapid technique for the determination of RNA and DNA in marine phytoplankton

An adaptation of the ethidium bromide technique for the analysisof nucleic acids is presented for marine phytoplankton. Themethod involves an initial homogenization of cells in phosphatebuffered saline, followed by incubation of subsamples of thecell homogenate in the presence and absence of ribonudease.Quantities of DNA and DNA + RNA in the respective sub-samplesare then determined by reaction with ethidium bromide. An evaluationis made of appropriate levels of bentonite required in the assayto inhibit endogenous cellular ribonucleases. Two nucleoproteindissociating agents, pronase and heparin, are also investigatedfor their capacities to enhance nucleic add fluorescence yield.The final recommended method resulted in maximum measured levelsof RNA and DNA in phytoplankton samples tested. The method canbe rapidly performed, involves a minimum amount of sample manipulation,and yields numbers having a high degree of precision. ¹Current address: School of Fisheries, University of Washington,WH-l0, Seattle, WA 98195, USA. ²Current address: Bigelow Laboratory for Ocean Sciences, WestBoothbay Harbor, ME 04575, USA.     

Effect of salinity on the distribution, growth, and toxicity of Karenia spp.

The first recorded bloom of Karenia spp., resulting in brevetoxin in oysters, in the low salinity waters of the Northern Gulf of Mexico (NGOMEX) occurred in November 1996. It raised questions about the salinity tolerance of Karenia spp., previously considered unlikely to occur at salinities <24 psu, and the likelihood that the bloom would reoccur in the NGOMEX. Salinity was investigated as a factor controlling Karenia spp. abundance in the field, using data from the NGOMEX 1996 bloom and Florida coastal waters from 1954 to 2004, and growth and toxin production in cultures of Karenia brevis (Davis) G. Hansen and Moestrup. During the NGOMEX bloom, Karenia spp. occurred much more frequently at low salinities than in Florida coastal waters over the last 50 years. The data suggest that the NGOMEX bloom started on the NW Florida Shelf, an area with a higher frequency of Karenia spp. at low salinities than the rest of Florida, and was transported by an unusual westward surface current caused by Tropical Storm Josephine. The minimum salinity at which growth occurred in culture ranged between 17.5 and 20 psu, but the optimal salinity ranged between low values of 20 or 25 and high values of 37.5–45 psu, depending on the clone. The effect of salinity on toxin production in one clone of K. brevis was complex, but at all salinities brevetoxin levels were highest during the stationary growth phase, suggesting that aging, high density blooms may pose the greatest public health threat. The results demonstrate that Karenia spp. can be a public health threat in low salinity areas, but the risk in the NGOMEX is relatively low. No bloom has occurred since the 1996 event, which was probably associated with a special set of conditions: a bloom along the Florida Panhandle and a tropical storm with a track that set up a westward current.     

Differences between adrenomedullary adrenaline and noradrenaline cells: quantitative electron-microscopic evaluation of their differential cellular association with supporting cells

Quantitative differences in cellular association of adrenomedullary chromaffin cells with other types of cells, mainly supporting cells, were studied. Adrenaline (A) and noradrenaline (NA) cells were compared. Electron micrographs (12000 x) of profiles of A and NA cells, bordering against other types of cells, were used for quantitative evaluation. Supporting cells constituted the majority of the non-chromaffin cell types. Occurrence frequencies of chromaffin cells contiguous with other types of cells were: (1) higher for A cells (68.9%, 199/289) than for NA cells (11.0%, 34/309) in case of small contact regions (²-test: P<0.001) and (2) higher for NA cells (68.3%, 211/309) than for A cells (9.7%, 28/289) in case of extended contact regions (P<0.001). In conclusion, the extent of cellular association with supporting cells was remarkably lower in A cells than in NA cells. Such an arrangement is likely to be appropriate for the extensive, homogeneous control and amplified response characteristic of A cells, and for the close range, complex control and more diverse responses characteristic of NA cells.Supported in part by The Karoji Memorial Fund for Medical Research in Hirosaki University, JapanThe authors, TK, TS and GT, wish to dedicate their part of this work to Dr. W.B. Quay on the occasion of his 65th birthday