Proteins drive genome compartmentalization across different length scales. While the identities of these proteins have been well-studied, the physical mechanisms that drive genome organization have remained largely elusive. Studying these mechanisms is challenging owing to a lack of methodologies to parametrize physical models in cellular contexts. Furthermore, because of the complex, entangled, and dense nature of chromatin, conventional live imaging approaches often lack the spatial resolution to dissect these principles. In this chapter, we will describe how to image the interactions of λ-DNA with proteins under purified and cytoplasmic conditions. First, we will outline how to prepare biotinylated DNA, functionalize coverslips with biotin-conjugated poly-ethylene glycol (PEG), and assemble DNA microchannels compatible for the imaging of protein-DNA interactions using total internal fluorescence microscopy. Then we will describe experimental methods to image protein-DNA interactions in vitro and DNA loop extrusion using Xenopus laevis egg extracts.

Although the genetic susceptibility to diseases has been extensively studied, the genetic loci and the primary molecular and cellular mechanisms that control disease tolerance are still largely unknown. Bovine paratuberculosis (PTB) is an enteritis caused by Mycobacterium avium subsp. paratuberculosis (MAP). PTB affects cattle worldwide and represents a major issue on animal health. In this study, the associations between host genetic and PTB tolerance were investigated using the genotypes from 277 Spanish Holstein cows with two distinct phenotypes: cases) infected animals with positive PCR and bacteriological culture results but without lesions in gut tissues (N= 24), and controls) animals with negative PCR and culture results but with PTB-associated lesions (N= 253). DNA from peripheral blood of the study population was genotyped with the Bovine EuroG MD Bead Chip, and the corresponding genotypes were imputed to whole-genome sequencing (WGS) data. A genome-wide association study was performed using the WGS data and the defined phenotypes in a case-control approach. A total of 142 single nucleotide polymorphisms (SNPs) were associated (false discovery rate ≤ 0.05, P values between 1.5 × 10-7 and 5.7 × 10-7) with tolerance (heritability= 0.55). The 40 SNPs with P-values < 5 × 10-7 defined 9 QTLs and 98 candidate genes located on BTA4, BTA9, BTA16, BTA25, and BTA26. Some of the QTLs identified in this study overlap with QTLs previously associated with PTB, bovine tuberculosis, mastitis, somatic cell score, bovine diarrhea virus persistent infection, tick resistance, and length of productive life. Two candidate genes with important roles in DNA damage response (ERCC4 and RMI2) were identified on BTA25. Functional analysis using the 98 candidate genes revealed a significant enrichment of the DNA packaging process (TNP2/PRMI1/PRM2/PRM3). In addition, the TNF-signaling (bta04668; TRAF5/CREB5/CASP7/CHUK) and the toxoplasmosis (bta05145; TGFβ2/CHUK/CIITA/SOCS1) pathways were significantly enriched. Interestingly, the nuclear Factor NF-κβ Inhibitor Kinase Alpha (CHUK), a key molecule in the regulation of the NF-κB pathway, was enriched in both pathways. Taken together, our results define a distinct immunogenetic profile in the PTB-tolerant animals designed to control bacterial growth, modulate inflammation, limit tissue damage and increase repair, thus reducing the severity of the disease.

Molecular motors that translocate DNA are ubiquitous in nature. During morphogenesis of double-stranded DNA bacteriophages, a molecular motor drives the viral genome inside a protein capsid. Several models have been proposed for the three-dimensional geometry of the packaged genome, but very little is known of the signature of the molecular packaging motor. For instance, biophysical experiments show that in some systems, DNA rotates during the packaging reaction, but most current biophysical models fail to incorporate this property. Furthermore, studies including rotation mechanisms have reached contradictory conclusions. In this study, we compare the geometrical signatures imposed by different possible mechanisms for the packaging motors: rotation, revolution, and rotation with revolution. We used a previously proposed kinetic Monte Carlo model of the motor, combined with Brownian dynamics simulations of DNA to simulate deterministic and stochastic motor models. We find that rotation is necessary for the accumulation of DNA writhe and for the chiral organization of the genome. We observe that although in the initial steps of the packaging reaction, the torsional strain of the genome is released by rotation of the molecule, in the later stages, it is released by the accumulation of writhe. We suggest that the molecular motor plays a key role in determining the final structure of the encapsidated genome in bacteriophages.

To date, the mechanisms responsible for radical change of chromatin structure in male germ cells during fertilization are unclear. Evidence suggesting the existence of proteolytic nuclear enzymes in mature human spermatozoids are presented in this work. The possible role of these previously unknown proteases in decondensation of chromatin of spermatozoids in a fertilized ovum is discussed. Application of the flow cytometry technique has shown that treatment of human spermatozoid nuclei with SH-reagents leads not only to destruction of disulfide bonds between protamine molecules that is necessary for their effective utilization but also induces specific endogenous proteolytic activity that consequently results in rather fast decondensation of chromatin followed by proteolytic cleavage of nuclear proteins. A chromatin decondensation process can be almost totally blocked by serine protease inhibitors and components of seminal fluid. An original cytochemical approach of binding of fluorescently labeled protease inhibitor to the target of investigation has been used in order to visualize the localization of proteases in male germ cell nuclei. The results of our study suggest that one of the factors of chromatin reorganization involved in the formation of male pronucleus is endogenous nuclear protease of spermatozoids, which is activated by glutathione or other SH-components of ovum cytoplasm.

A murine cytomegalovirus (MCMV) temperature-sensitive (ts) mutant, tsm5, of the K181 (Birmingham) strain, showed approximately 10-fold and approximately 10,000-fold reductions in yields at the permissive (33 degrees C) and non-permissive temperature (40 degrees C), respectively. It did not replicate to detectable levels in any tissue of 1-week-old Balb/c mice for up to 21 days following i.p. inoculation with 4 x 10(3) pfu although it did replicate, albeit with considerably delayed kinetics, in SCID mice. tsm5 expressed all kinetic classes of transcript (immediate-early, early and late) both in vitro at the non-permissive temperature and in vivo. To identify mutations contributing to this phenotype, chimaeric viruses produced from overlapping cosmids generated from tsm5 and the Smith strain of MCMV were examined. A virus, Smith/tsm5DGIK, comprising the central conserved region of the tsm5 genome, was not attenuated at 33 or 37 degrees C but was ts at 40 degrees C, although not to the same extent as tsm5. In contrast to tsm5, this chimaeric virus replicated to similar levels as parental viruses in adult BALB/c mice. These results suggested that genes contributing to reduced replication at 33 degrees C and lack of replication in vivo are located at the ends of the tsm5 genome while those contributing to the ts phenotype are located in the central conserved region of the genome. Sequencing of some immune evasion genes known to be located at the 3\' or 5\' ends of the MCMV genome showed that no mutations were present in ORFs m04, m06, M33, M37, m38.5, m144, m152, or m157 although mutations were found in M27 (A658S) and M36Ex1 (V54I). tsm5 made few capsids at 40 degrees C and these lacked DNA. DNA synthesis was significantly reduced in tsm5-infected cells at 40 degrees C although DNA cleavage occurred with close to wt efficiency. Sequencing of the herpesvirus conserved cis-acting elements, pac1 and pac2, and genes involved in DNA packaging and cleavage located in the central core region of the genome identified few point mutations. Two were identified that alter the encoded protein in tsm5 ORFs M98 (P324S) and M56 (G439R). Furthermore, a point mutation (C890Y) was identified in M70, the primase. Another mutant, tsm30, which is also defective in DNA packaging and processing, has a point mutation in M52 (D494N). Thus, a number of mutations have been identified in tsm5 that suggests that it is defective in genes involved in immune evasion, DNA replication and DNA encapsidation.

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