Initiation of replication in the Chinese hamster dihydrofolate reductase domain

Two-dimensional (2-D) gel analysis of replication intermediates in the Chinese hamster dihydrofolate reductase domain has suggested that nascent chains can initiate at any of a large number of sites scattered throughout a ～50 kb “initiation locus” (although the level of initiation detected at any given site within this region was relatively low). This result contrasts markedly with data from anin vitro strand switching assay suggesting that >80% of initiations occur within a single 500 bp fragment lying within the initiation locus. In an effort to reconcile these two disparate views of the initiation reaction, we have questioned the validity of our 2-D gel data in several ways. We show here that: 1) the number of replication bubbles detected in the DHFR locus in the early S period is markedly increased when the cells are released from a synchronizing agent that inhibits initiationper se, rather than from aphidicolin, which is a chain elongation inhibitor; 2) initiation in the DHFR domain occurs only during the first 90 min of the S period, as would be expected of an early-firing origin; 3) a pulse of³H-thymidine moves through the structures observed on 2-D gels with the kinetics expected ofbonafide replication intermediates; and 4) preparations of replication intermediates that are subsequently analyzed on 2-D gels appear, by electron microscopy, to represent the typical theta structures and single-forked molecules expected of bidirectional origins of replication; no unusual structures (e.g., microbubbles) were seen.     

Murine p53 inhibits the function but not the formation of SV40 T antigen hexamers and stimulates T antigen RNA helicase activity

We have characterized the effects of p53 on several biochemical activities of simian virus 40 (SV40) large tumor (T) antigen. While p53 induced a strong inhibition of the T antigen DNA helicase activity, surprisingly, its RNA helicase activity was stimulated. This supports the liklihood that the DNA and RNA helicase activities of T antigen reflect discrete functions. p53 did not significantly affect the ATP-dependent conversion of T antigen monomers to hexamers. However, the ability of these hexamers to assemble on a DNA fragment containing the viral origin was impaired by p53. Thus, these results suggest that p53 inhibits the function but not the formation of T antigen multimers. This conclusion was further supported by the observation that the addition of a purified p53:T antigen complex was as inhihitory as free p53 to the DNA helicase activity of free T antigen. Thus our data indicates that the targets of p53 inhibition are the functional units of T antigen, namely the hexamers.     

The 3′→5′ exonuclease associated with HeLa DNA polymerase epsilon

The 3′→5′ exonuclease activity of highly purified large form of human DNA polymerase epsilon was studied. The activity removes mononucleotides from the 3′ end of an oligonucleotide with a non-processive mechanism and leaves 5′-terminal trinucleotide non-hydrolyzed. This is the case both with single-stranded oligonucleotides and with oligonucleotides annealed to complementary regions of M13DNA. However, the reaction rates with single-stranded oligonucleotides are at least ten-fold when compared to those with completely base-paired oligonucleotides. Conceivably, mismatched 3′ end of an oligonucleotide annealed to M13DNA is rapidly removed and the hydrolysis is slown down when double-stranded region is reached. The preferential removal of a non-complementary 3′ end and the non-processive mechanism are consistent with anticipated proofreading function. In addition to the 3′→5′ exonuclease activity, an 5′→3′ exonuclease activity is often present even in relatively highly purified DNA polymerase epsilon preparates suggesting that such an activity may be an essential com-ponent for the action of this enzymein vivo. Contrary to the 3′→5′ exonuclease activity, the 5′→3′ exonuclease is separable from the polymerase activity.     

Heat aggregation studies of phycobilisomes, ferritin, insulin, and immunoglobulin by dynamic light scattering.

Dynamic laser light scattering studies on the heat aggregation behavior of phycobilisomes (PBS), ferritin, insulin, and immunoglobulin (IgG) in dilute aqueous solutions has been reported. Except for PBS, results are reported for heat aggregation trends in these proteins for three different pH environments (4.0, 7.5, 9.1). For PBS, studies were performed only in the neutral buffer medium (pH 7.5). The experiments were performed in the very dilute concentration regime (between 0.23 and 1.8 gL-1). For all these samples heat aggregation and dissociation trends were found to be linear with temperature. Upon temperature reversal (self-cooling), hysteresis-like behavior observed in insulin was found to be predominantly large at pH 7.5. PBS, ferritin, and IgG showed no such behavior at any of three pH values, and retraced their path of aggregation while dissociating on temperature reversal. Heat aggregation and dissociation processes in ferritin were found to be independent of pH. The IgG samples showed smooth aggregation tendency only up to 35 degrees C in the buffer media pH 4.0 and 9.1, whereas for pH 7.0 the same could be observed until 60 degrees C. Low polydispersity in the correlation spectra was observed in case of all these samples.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Expression of anchorin CII mRNA by cultured chondrocytes

The establishment of a cell culture system promoting chondrocyte differentiation has been utilized to better characterize phenotypic stages of chondrogenesis at the cellular level. Although the expression of the type II collagen gene has been studied during “in vitro” chondrocyte differentiation, little is known about the expression of the gene coding for its receptor: anchorin CII. The modulation of the anchorin mRNA steady state level in chick embryo chondrocytes at different developmental stages is described here.The anchorin mRNA level was low in dedifferentiated chondrocytes, progressively increased after the cell transfer into suspension (a condition promoting differentiation), reached its maximal value after 4 weeks and decreased after 5 weeks.Therefore anchorin CII mRNA reaches its maximum level in hypertrophic stage II chondrocytes.     

Molecular Evidence and the Origin and Development of the Domesticated Sunflower (<Emphasis Type="Italic">Helianthus annum</Emphasis>, Asteraceae)

The domesticated sunflower,Helianthus annuus, is an important economic crop, yet molecular data regarding its evolution are limited. Here we review morphological, geographical, archaeological, and molecular evidence pertaining to its origin and development. New isozyme and chloroplast DNA (cpDNA) evidence is also presented.Morphological, geographical, and archaeological evidence has led to the hypothesis that the domesticated sunflower was derived from a wild/weedy form ofH. annuus possibly in the Midwest. Molecular evidence was concordant with this hypothesis. A high degree of enzymatic and cpDNA sequence similarity was observed between wild and domesticatedH. annuus, and domesticatedH. annuus contained a subset of the alleles and cpDNAs found in wildH. annuus. The extensive polymorphism in the wild plants and the virtual monomorphism in cultivated lines for both isozyme and cpDNA phenotypes further suggest a single origin of the domesticated sunflower from a very limited gene pool. In addition, Native American varieties of the domesticated sunflower were genetically more variable than other cultivated lines, possibly indicating that they gave rise to the other cultivated stocks. Molecular evidence did not, however, allow conclusions as to the exact geographic origin of the domesticated sunflower.     

Exogenous glycosaminoglycans (GAG) differentially modulate GAG synthesis by anchorage-independent cultures of the outer cells from neonatal rat calvaria in the absence and presence of TGF-β

In anchorage-dependent (AD) cultures of the outer cell population (OCP) from neonatal rat calvaria, transforming growth factor-₁ (TGF-) specifically upregulated the synthesis of chondroitin sulfate (CS) proteoglycan (PG) and uncoupled the inhibitory effect of increasing cell density on CS PG synthesis (reference #30). Utilizing the same cell population, we have further examined the possibility that glycosaminoglycans (GAG) known to be synthesized and secreted by bone cells might exert feedback effects on GAG synthesis and/or its stimulation by TGF-. Although addition of TGF- alone stimulated net synthesis of HA and CS in both AD and anchorage-independent (AI) cultures, significant alterations of basal and TGF--stimulated GAG synthesis by exogenous GAGs were observed only in AI cultures. In AI cultures exogenously added hyaluronic acid (HA) markedly enhanced the basal synthesis of HA and CS while heparin (H) suppressed the basal synthesis of HA, CS as well as dermatan sulfate (DS). Also, the addition of HA markedly potentiated the stimulation by TGF- of HA and CS synthesis as did heparan sulfate (HS) for CS and DS synthesis. H suppressed the stimulation of the synthesis of HA, CS and DS by TGF-. Overall, our results indicate specific effects of individual GAGs on basal and TGF--stimulated GAG synthesis in OCP cultures. We suggest that some of the GAGs in the OCP microenvironment (which with the exception of HA are covalently linked to protein cores of secreted PGs), acting in concert with TGF-, may serve as an amplification system for upregulating GAG synthesis in the rapidly growing neonatal calvarium.     

Estimation of the heritability of hair and eye color

In the present study the colours of hair are regarded as quantitative genetic characters. The genetic and environmental components of the total variance of the characters are determined by analysis of correlation between parents and children as well as full sibs. An analysis of correlation coefficients makes it possible to further examine whether autosomal dominance or X-linked factors play a role in the inheritance. The correlations between the parents make the genetical analysis more complicated as they raise the correlation coefficients between relatives. The estimates of heritability are 0.61 (hair-colour) and 0.80 (eye-colour). These estimates may serve as preliminary values of orientation. Only further studies will show whether the relative large part of the variance caused by the environment can be confirmed. Dominance may play a small role in the case of hair colour, while it seems to be absent in eye-colour. There are no sufficient indications of X-linked factors for both characters.