Replication of bacteriophage lambda DNA. Examination of variants containing double origins and observation of a bias in directionality

Bacteriophage λ variants have been constructed that possess two λ ori sites. Replicative intermediates resulting from infection with these phages have been investigated. We find that initiation of replication from the ori site on an EcoRI fragment (containing all the DNA sequences from within the red gene to the middle of gene O) cloned in the inverted orientation is predominantly bidirectional but occurs at a decreased frequency. Double initiations were observed at low frequency. However, a second cloned ori fragment (carrying two large deletions and a small insertion) cloned in the normal orientation demonstrated insignificant levels of replication from the cloned site unless the normal ori had already initiated.A bias in directionality of λ replication has been observed. Molecules that replicate unidirectionally propagate to the right more often than to the left. If the cloned ori-containing EcoRI fragment is inserted with reversed polarity, then the bias is towards the left. Bidirectional λ replicative intermediates also appear to show a similar bias but this is superimposed on a large, apparently random, effect that results in asymmetric growing-point propagation.     

Effects of butylated hydroxyanisole on the aryl hydrocarbon hydroxylase of rats and mice

The effects of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) on the aryl hydrocarbon hydroxylase (AHH) activities in the liver, lung and skin of rats and mice have been studied to examine the possible mechanisms of the anticarcinogenic actions of these compounds. Both compounds inhibit the hydroxylase activities of hepatic microsomes and nuclei, with BHA a more potent inhibitor than BHT. The AHH of lung microsomes is inhibited to a lesser extent by BHA and BHT than that of the liver. The AHH activities of both liver and lung microsomes become less susceptible to the inhibition after pretreatment of the animals with 3-methylcholanthrene (MC) or 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) but phenobarbital (PB) pretreatment does not produce such an effect. In skin homogenates, however, the AHH activities of control rats and mice are not inhibited by BHA and BHT. The only skin sample which is inhibited by BHA and BHT is that from TCDD-pretreated mice. It has been established that the extent of inhibition with different samples is related to the concentration of BHA in the incubation but not to the amounts or specific activities of microsomes used. Double reciprocal plots suggest that BHA exerts a mixed inhibition on the hydroxylase of liver microsomes with a K_i of 7.7 μM. Analysis of the metabolites of benzo[a]pyrene (BP) shows that BHA inhibits the formation of various metabolites uniformly without changing the regio-selectivity of the enzyme system. The mechanism of inhibition has also been studied with a reconstituted AHH system consisting of cytochrome P-450 (P-450), reductase and phospholipid. The system with P-450 isolated from PB-induced microsomes is inhibited to a much greater extent than that with MC-induced P-450. The results indicate that the inhibitory action of BHA is dependent on the species of the animal, tissue types and treatment with inducers.     

Specificity of Insertion by the Translocatable Tetracycline-Resistance Element Tn10

Genetic analysis of 131 independent transpositions of the tetracycline-resistance element Tn10 from a single site in phage P22 into the histidine operon of Salmonella typhimurium reveals that Tn10 insertions are not randomly distributed along this chromosomal target. The insertions occur in 22 different "clusters"; insertions within each cluster are very tightly linked in recombination tests. Tn10 insertions are not evenly distributed among the identified clusters. The existence of these clusters suggests that this chromosomal target contains particular genetic signals that guide Tn10 to particular preferred positions for insertion. Insertions within each cluster occur in both orientations with roughly equal frequency.--The relationship among different insertions within each cluster has been examined. The resolution of genetic mapping places an upper limit of about 50 basepairs on the distance between different insertions within a cluster. Different insertions within a cluster usually have the same reversion frequency; however, heterogeneity in reversion frequency has been detected in at least two clusters. For most clusters, the available data are consistent with the simple possibility that all insertions within a cluster are at identical positions; however, the data do not exclude other possibilities.     

Transdetermination of Drosophila imaginal discs cultured in vitro

Imaginal discs of Drosophila melanogaster undergo transdetermination when cultured in vivo in the abdominal cavity of adult female hosts. We report here that leg discs cultured in vitro, in a recently developed system, also undergo transdetermination. Whether cultured in vivo or in vitro, leg discs produce a similar range of specific transdetermined structures. Moreover, in comparison to discs cultured in vivo, the discs cultured in vitro exhibit a similar correlation between the amount of growth and the total frequency of transdetermination.     

Protoplast induction inMicrasterias andCosmarium

Summary Protoplasts of the desmidsMicrasterias angulosa, M. denticulata, M. thomasiana andCosmarium turpinii were obtained by incubating cells in Waris' liquid medium + 0.3 M mannitol + 2% Cellulysin for 1–3 hours. One osmotically fragile protoplast was formed at the isthmus from the joint contents of both semicells. The resultant protoplasts were bright green and remained so for more than 5 days in the osmotically protective medium. The protoplast yield was better than 80%. The empty cell walls were not digested by the Cellulysin or by autolytic enzymes.     

The zinc content of yeast alcohol dehydrogenase

Analyses for zinc in high specific activity preparations of yeast alcohol dehydrogenase (YADH) indicate a metal content of 1.8–1.9 moles of zinc per mole of enzyme subunit. This zinc content is observed for YADH prepared from Bakers yeast by recrystallization from Am₂SO₄ containing 1 mM EDTA, followed by chromatography on DE-52 and Sephadex-G-200. YADH obtained from Boehringer-Mannheim is characterized by a variable specific activity: preparations with Sp. Ac. = 380–400 U/mg contain 1.8–1.9 moles of zinc per mole of subunit. Dialysis of YADH against EDTA (pH 8.5, 25°, under N₂) reduces the specific activity and zinc content in an approximately linear fashion down to a Sp. Ac. = 150 U/mg, consistent with the preferential loss of a single, weakly bound zinc per subunit which is essential for catalytic activity. Dialysis of YADH against 1 mM ZnCl₂ (pH 6.5–8.5, 25°, under N₂) does not lead to an increase in the zinc content of the enzyme, indicating that under these conditions zinc does not bind adventitiously to YADH. Dialysis against 50 mM CoSO₄ (pH 5.5, 25°, under N2, 60–90 hr) leads to an exchange of ≈ 40% of the enzyme-bound zinc by cobalt. Our preparations of YADH are consistently characterized by a zinc content of ≈ 2 per subunit and we are unable to reduce the zinc content of YADH by dialysis against EDTA without a concomitant loss in enzyme activity, in contrast to reports of one zinc per subunit [Veillon, C. and Sytkowski, A.J., BBRC $\text{67}$: 1499 (1975); Vallee, B.L. and Hoch, F.L., Proc. Nat. Acad. Sci. USA $\text{41}$: 327 (1955)]. The findings reported here, together with the observed structural similarities between YADH and horse liver alcohol dehydrogenase [Jornvall, H., Woenckhaus, C. and Johnscher, G., Eur. J. Biochem. $\text{53}$: 71 (1975)], suggest a role for zinc at both a structural and catalytic site in YADH.     

Using genetic variation to disentangle the complex relationship between food intake and health outcomes

Diet is considered as one of the most important modifiable factors influencing human health, but efforts to identify foods or dietary patterns associated with health outcomes often suffer from biases, confounding, and reverse causation. Applying Mendelian randomization in this context may provide evidence to strengthen causality in nutrition research. To this end, we first identified 283 genetic markers associated with dietary intake in 445,779 UK Biobank participants. We then converted these associations into direct genetic effects on food exposures by adjusting them for effects mediated via other traits. The SNPs which did not show evidence of mediation were then used for MR, assessing the association between genetically predicted food choices and other risk factors, health outcomes. We show that using all associated SNPs without omitting those which show evidence of mediation, leads to biases in downstream analyses (genetic correlations, causal inference), similar to those present in observational studies. However, MR analyses using SNPs which have only a direct effect on the exposure on food exposures provided unequivocal evidence of causal associations between specific eating patterns and obesity, blood lipid status, and several other risk factors and health outcomes.     

nAdder: A scale-space approach for the 3D analysis of neuronal traces

Tridimensional microscopy and algorithms for automated segmentation and tracing are revolutionizing neuroscience through the generation of growing libraries of neuron reconstructions. Innovative computational methods are needed to analyze these neuronal traces. In particular, means to characterize the geometric properties of traced neurites along their trajectory have been lacking. Here, we propose a local tridimensional (3D) scale metric derived from differential geometry, measuring for each point of a curve the characteristic length where it is fully 3D as opposed to being embedded in a 2D plane or 1D line. The larger this metric is and the more complex the local 3D loops and turns of the curve are. Available through the GeNePy3D open-source Python quantitative geometry library (https://genepy3d.gitlab.io), this approach termed nAdder offers new means of describing and comparing axonal and dendritic arbors. We validate this metric on simulated and real traces. By reanalysing a published zebrafish larva whole brain dataset, we show its ability to characterize different population of commissural axons, distinguish afferent connections to a target region and differentiate portions of axons and dendrites according to their behavior, shedding new light on the stereotypical nature of neurites’ local geometry.     

SARS-CoV-2-specific T cells associate with inflammation and reduced lung function in pulmonary post-acute sequalae of SARS-CoV-2

As of January 2022, at least 60 million individuals are estimated to develop post-acute sequelae of SARS-CoV-2 (PASC) after infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). While elevated levels of SARS-CoV-2-specific T cells have been observed in non-specific PASC, little is known about their impact on pulmonary function which is compromised in the majority of these individuals. This study compares frequencies of SARS-CoV-2-specific T cells and inflammatory markers with lung function in participants with pulmonary PASC and resolved COVID-19 (RC). Compared to RC, participants with respiratory PASC had between 6- and 105-fold higher frequencies of IFN-γ- and TNF-α-producing SARS-CoV-2-specific CD4⁺ and CD8⁺ T cells in peripheral blood, and elevated levels of plasma CRP and IL-6. Importantly, in PASC participants the frequency of TNF-α-producing SARS-CoV-2-specific CD4⁺ and CD8⁺ T cells, which exhibited the highest levels of Ki67 indicating they were activity dividing, correlated positively with plasma IL-6 and negatively with measures of lung function, including forced expiratory volume in one second (FEV¹), while increased frequencies of IFN-γ-producing SARS-CoV-2-specific T cells associated with prolonged dyspnea. Statistical analyses stratified by age, number of comorbidities and hospitalization status demonstrated that none of these factors affect differences in the frequency of SARS-CoV-2 T cells and plasma IL-6 levels measured between PASC and RC cohorts. Taken together, these findings demonstrate elevated frequencies of SARS-CoV-2-specific T cells in individuals with pulmonary PASC are associated with increased systemic inflammation and decreased lung function, suggesting that SARS-CoV-2-specific T cells contribute to lingering pulmonary symptoms. These findings also provide mechanistic insight on the pathophysiology of PASC that can inform development of potential treatments to reduce symptom burden.     

TBX2 acts as a potent transcriptional silencer of tumour suppressor genes through interaction with the CoREST complex to sustain the proliferation of breast cancers

Chromosome 17q23 amplification occurs in 20% of primary breast tumours and is associated with poor outcome. The TBX2 gene is located on 17q23 and is often over-expressed in this breast tumour subset. TBX2 is an anti-senescence gene, promoting cell growth and survival through repression of Tumour Suppressor Genes (TSGs), such as NDRG1 and CST6. Previously we found that TBX2 cooperates with the PRC2 complex to repress several TSGs, and that PRC2 inhibition restored NDRG1 expression to impede cellular proliferation. Here, we now identify CoREST proteins, LSD1 and ZNF217, as novel interactors of TBX2. Genetic or pharmacological targeting of CoREST emulated TBX2 loss, inducing NDRG1 expression and abolishing breast cancer growth in vitro and in vivo. Furthermore, we uncover that TBX2/CoREST targeting of NDRG1 is achieved by recruitment of TBX2 to the NDRG1 promoter by Sp1, the abolishment of which resulted in NDRG1 upregulation and diminished cancer cell proliferation. Through ChIP-seq we reveal that 30% of TBX2-bound promoters are shared with ZNF217 and identify novel targets repressed by TBX2/CoREST; of these targets a lncRNA, LINC00111, behaves as a negative regulator of cell proliferation. Overall, these data indicate that inhibition of CoREST proteins represents a promising therapeutic intervention for TBX2-addicted breast tumours.