Immunocytochemical localization of transient DNA strand breaks in differentiating myotubes using in situ nick-translation

We have localized DNA strand breaks during in vitro chicken myogenesis by repairing nicks in nuclei of fixed cell monolayers in situ with biotin-11-dUTP, followed by immunocytochemical detection of incorporated biotin with rabbit anti-biotin and FITC-labeled goat anti-rabbit antibodies. No accumulations of biotin sufficient for immunocytochemical detection were observed in 23-hr cultures of dividing cells. In 33- and 43-hr cultures, biotin was first detected in only 3% of the nuclei, all of which appeared to be in fusing myoblasts or small myotubes. In contrast, cultures of young, highly fused myotubes (56 hr) exhibited 18% biotinylated nuclei; virtually all of these nuclei, most of which were grouped as aggregates, were within myotubes. In older cultures (73 and 94 hr) incorporation of biotin into myotube nuclei markedly decreased, while increases were noted in nuclei of mononuclear cells. These results indicate that extensive single-stranded DNA nicking occurs in nuclei of young myotubes, followed by repair as terminal differentiation ensues.     

Spermine-induced phosphorylation of myotube histones by endogenous nuclear protein kinases

The effects of spermine on phosphorylation of nuclear proteins in isolated nuclei from proliferation and myotube stage cells during differentiation of cultured chicken myoblasts have been investigated. Incorporation of phosphate from 32P-gamma-ATP was assessed by incubating nuclei with and without 2 mM spermine, which caused an approx. 1.5-fold increase in phosphorylation of total nuclear proteins in both cell types. Modification of individual proteins was assessed by extracting basic proteins in dilute acid, followed by SDS-electrophoresis on 18% acrylamide gels and radioautography. Results indicated that whereas most phosphoproteins in both cell types were increased 1.5-2.0-fold, phosphorylation of a 31 000 D band increased several-fold. Most strikingly, myotube nuclei displayed selective 3.5- and 9-fold increases in specific radioactivity of histones Hla and H3, respectively, which normally exhibit little, if any, phosphorylation.     

Affinity isolation of active murine erythroleukemia cell chromatin: uniform distribution of ubiquitinated histone H2A between active and inactive fractions.

This laboratory recently reported the development of a biotin-cellulose/streptavidin affinity chromatography method based on the DNase I sensitivity of active chromatin to isolate a DNA fraction from murine erythroleukemia (MEL) cells that is more than 15-fold enriched in active genes (Dawson et al.: Journal of Biological Chemistry 264:12830-12837, 1989). We now report the extension of this technique to isolate and characterize chromatin that is enriched in active genes. In this approach, DNA in nuclei isolated from MEL cells was nicked with DNase I at a concentration that does not digest the active beta-globin gene, followed by repair of the nicks with a cleavable biotinylated nucleotide analog, 5-[(N-biotin-amido)hexanoamido-ethyl-1,3'-dithiopropionyl-3- aminoallyl]-2'- deoxyuridine 5'-triphosphate (Bio-19-SS-dUTP), during a nick-translation reaction. After shearing and sonication of the nuclei to solubilize chromatin, chromatin fragments containing biotin were separated from non-biotinylated fragments by sequential binding to streptavidin and biotin cellulose. The bound complex contained approximately 10% of the bulk DNA. Reduction of the disulfide bond in the biotinylated nucleotide eluted approximately one-half of the affinity isolated chromatin. Hybridization analysis of DNA revealed that whereas inactive albumin sequences were equally distributed among the chromatin fractions, virtually all of the active beta-globin sequences were associated with chromatin fragments which had bound to the affinity complex. Western blot assessment for ubiquitinate histones revealed that ubiquitinated histone H2A (uH2A) was uniformly distributed among active (bound) and inactive (unbound) chromatin fractions.     

Historical mortality in massive<Emphasis Type="Italic"> Porites</Emphasis> from the central Great Barrier Reef,Australia: evidence for past environmental stress?

>Two hiatuses in coral skeleton growth, associated tissue death and subsequent regrowth, were discovered while dating eight multi-century Porites coral cores collected from the central Great Barrier Reef (GBR), Australia. Cross-dating of characteristic annual luminescent lines visible in the coral core slices under UV-light (Hendy et al. 2003) accurately dated the two events to 1782–85 and 1817 a.d.. Die-off scars were observed in only one core for each event. X-radiographs and photographs taken under UV-light show the pattern of regrowth and the period taken by the coral to recover. Bioerosion, predominately by boring sponges ( Cliona spp.), of the exposed coral surface following the 1782–85 event caused a hiatus of up to 14 years' growth, with the coral taking 7–8 years to reclaim the whole surface contained within the 9-cm-diameter core. Contemporary historical and proxy-climate records indicate that El Niño climatic conditions occurred at the time of both growth discontinuities. Intense luminescence observed in corals growing continuously during the 1817 event suggests that low salinity from river runoff was a contributing factor, analogous environmental conditions to those that were associated with the 1998 bleaching event in the GBR.     

Cell-to-cell movement of potexviruses: evidence for a ribonucleoprotein complex involving the coat protein and first triple gene block protein

The triple gene block proteins (TGBp1-3) and coat protein (CP) of potexviruses are required for cell-to-cell movement. Separate models have been proposed for intercellular movement of two of these viruses, transport of intact virions, or a ribonucleoprotein complex (RNP) comprising genomic RNA, TGBp1, and the CP. At issue therefore, is the form(s) in which RNA transport occurs and the roles of TGBp1-3 and the CP in movement. Evidence is presented that, based on microprojectile bombardment studies, TGBp1 and the CP, but not TGBp2 or TGBp3, are co-translocated between cells with viral RNA. In addition, cell-to-cell movement and encapsidation functions of the CP were shown to be separable, and the rate-limiting factor of potexvirus movement was shown not to be virion accumulation, but rather, the presence of TGBp1-3 and the CP in the infected cell. These findings are consistent with a common mode of transport for potexviruses, involving a non-virion RNP, and show that TGBp1 is the movement protein, whereas TGBp2 and TGBp3 are either involved in intracellular transport or interact with the cellular machinery/docking sites at the plasmodesmata.     

Environmental controls on growth of the massive coral Porites

Annual density banding provided growth characteristics for 245 similar-sized, massive colonies of Porites from similar locations on 29 reefs from across the length and breadth of the Great Barrier Reef (GBR), Australia. Values obtained were density, extension rate, and calcification rate. Tissue thickness, the depth to which skeletons were occupied by tissue at the time of collection, was also measured. Extension rate, calcification rate, and tissue thickness were significantly greater at the top of colonies than at the sides. Extension rate and calcification rate decreased from north to south along the GBR (latitudinal range of approximately 9 degrees ) and were significantly and directly related to annual average sea surface temperature (SST; range approximately 25-27 degrees C). For each 1 degrees C rise in SST, average annual calcification increased by 0.39 g cm(-2) year(-1) and average annual extension increased by 3.1 mm year(-1) (c.f. average values of 1.63 g cm(-2) year(-1) and 12.9 mm year(-1), respectively). Density was inversely correlated with extension rate and increased with distance offshore. Data for massive Porites colonies from the GBR were extended though 20 degrees of latitude and an average annual SST range of 23-29 degrees C using published data for the Hawaiian Archipelago (Grigg, R.W., 1981. Coral reef development at high latitudes in Hawaii. Proc. 4th Int. Coral Reef Symp., Manila, Vol. 1, pp. 687-693; Grigg, R.W., 1997. Paleoceanography of coral reefs in the Hawaiian-Emperor Chain - revisited. Coral Reefs 16, S33-S38) and Phuket, Thailand (Scoffin. T.P., Tudhope. A.W., Brown. B.E., Chansang. H., Cheeney. R.F., 1992. Patterns and possible environmental controls of skeletogenesis of Porites lutea, South Thailand. Coral Reefs 11, 1-11). The response of calcification rate to temperature remained linear. Variation in annual average SST accounted for 84% of the variance. For each 1 degrees C rise in SST, average annual calcification increased by 0.33 g cm(-2) year(-1) and average annual extension increased by 3.1 mm year(-1) (c.f. average values of 1.50 g cm(-2) year(-1) and 11.6 mm year(-1), respectively). The sensitivity of calcification rate in Porites to SST, combined with observed 20th Century increases in SSTs, suggests that calcification rates may have already significantly increased along the GBR in response to global climate change.     

Comparative analysis of the plasmids from two isolates of "Candidatus Phytoplasma australiense"

Two plasmids from the plant-pathogenic mollicute "Candidatus Phytoplasma australiense" were completely sequenced from two isolates derived from different plant hosts. Plasmid pPAPh2 (3607bp) was obtained from Phormium showing Phormium yellow leaf symptoms and pPASb11 (3635bp) from strawberry showing strawberry lethal yellows symptoms. The plasmids varied in their copy number and nucleotide sequence yet contained the same four open reading frames (ORFs). The deduced amino acid sequence derived from ORF1 shares similarity with hypothetical proteins encoded on the plasmids from onion yellows and beet leafhopper-transmitted virescence agent phytoplasmas. The deduced amino acid sequences of both ORF2 and ORF3 share similarity with functionally unknown proteins on the chromosome of onion yellows phytoplasma. An ORF with a similar sequence to ORF2 is also present on the chromosome of "Ca. P. australiense." The deduced amino acid sequence derived from ORF4 is most similar to replication proteins encoded by other phytoplasma plasmids and by geminiviruses, the only protein on the plasmids for which a putative function can be assigned. The identities of the deduced amino acid sequences of ORF1, ORF2, ORF3, and ORF4 between pPAPh2 and pPASb11 were 89, 68, 91, and 68%, respectively; the differences being consistent with the subgroup status of the parental phytoplasmas.     

The baubellum is more developmentally and evolutionarily labile than the baculum

Understanding the evolutionary forces that influence sexual dimorphism is a fundamental goal in biology. Here, we focus on one particularly extreme example of sexual dimorphism. Many mammal species possess a bone in their penis called a baculum. The female equivalent of this bone is called the baubellum and occurs in the clitoris, which is developmentally homologous to the male penis. To understand the potential linkage between these two structures, we scored baculum/baubellum presence/absence across 163 species and analyzed their distribution in a phylogenetic framework. The majority of species (N = 134) shared the same state in males and females (both baculum and baubellum present or absent). However, the baubellum has experienced significantly more transitions, and more recent transitions, so that the remaining 29 species have a baculum but not a well‐developed baubellum. Even in species where both bones are present, the baubellum shows more ontogenetic variability and harbors more morphological variation than the baculum. Our study demonstrates that the baculum and baubellum are generally correlated across mammals, but that the baubellum is more evolutionarily and developmentally labile than the baculum. The accumulation of more evolutionary transitions, especially losses in the baubellum, as well as noisier developmental patterns, suggests that the baubellum may be nonfunctional, and lost over time.     

Porites growth characteristics in a changed environment: Misima Island, Papua New Guinea

 The construction in 1988 of an open-cut gold mine and ore-processing facility at Misima Island, Papua New Guinea, resulted in disturbance of the adjacent fringing coral reef, mostly because of large increases in sedimentation. This provided an opportunity to examine whether growth characteristics of the major reef-building coral, Porites, changed in response to this sudden and sustained increase in sedimentation. Annual variation in skeletal density was measured in 93 colonies variously affected by sedimentation. The colonies provided data for average annual density, annual extension and annual calcification covering the periods 5 y before and 5 y after mining operations began. The average depth of skeleton occupied by tissue (tissue layer thickness) was also measured for most colonies. There was high mortality of Porites in regions strongly affected by increased sedimentation. In colonies that survived, density, extension and calcification tended to be less (in some cases significantly) in the period after mining operations began compared with pre-construction levels. However, these decreases were not linked with proximity to the mine site and probably reflect a regional-scale response of Porites growth to some other environmental change. This suggests that periods of high sedimentation may not be recorded by the growth characteristics of massive Porites. Average growth characteristics of surviving Porites from Misima Island were similar to those from inshore reefs of the northern Great Barrier Reef (GBR). Tissue layer thickness in Porites from the control areas at Misima Island were also similar to colonies from the northern inshore GBR reefs. However, tissue layer thickness significantly decreased with increased proximity to the mine site at Misima Island. Accepted: 15 May 1999