Summary
Chromosome rearrangements and the way that they impact genetic differentiation and speciation have long raised questions from evolutionary biologists. They are also a major concern for ...breeders because of their bearing on chromosome recombination. Banana is a major crop that derives from inter(sub)specific hybridizations between various once geographically isolated Musa species and subspecies. We sequenced 155 accessions, including banana cultivars and representatives of Musa diversity, and genotyped‐by‐sequencing 1059 individuals from 11 progenies. We precisely characterized six large reciprocal translocations and showed that they emerged in different (sub)species of Musa acuminata, the main contributor to currently cultivated bananas. Most diploid and triploid cultivars analyzed were structurally heterozygous for 1 to 4 M. acuminata translocations, highlighting their complex origin. We showed that all translocations induced a recombination reduction of variable intensity and extent depending on the translocations, involving only the breakpoint regions, a chromosome arm, or an entire chromosome. The translocated chromosomes were found preferentially transmitted in many cases. We explore and discuss the possible mechanisms involved in this preferential transmission and its impact on translocation colonization.
Significance Statement
Chromosome rearrangements, the way that they emerged and their potential impact on speciation and breeding raise important questions. We report on the characterization of several large reciprocal translocations in banana, Musa acuminata, identify the genetic group in which they emerged, characterize their impacts on chromosome recombination, and explore possible mechanisms allowing their colonization.
The Sec61 complex forms a protein-conducting channel in the endoplasmic reticulum membrane that is required for secretion of soluble proteins and production of many membrane proteins. Several natural ...and synthetic small molecules specifically inhibit Sec61, generating cellular effects that are useful for therapeutic purposes, but their inhibitory mechanisms remain unclear. Here we present near-atomic-resolution structures of human Sec61 inhibited by a comprehensive panel of structurally distinct small molecules-cotransin, decatransin, apratoxin, ipomoeassin, mycolactone, cyclotriazadisulfonamide and eeyarestatin. All inhibitors bind to a common lipid-exposed pocket formed by the partially open lateral gate and plug domain of Sec61. Mutations conferring resistance to the inhibitors are clustered at this binding pocket. The structures indicate that Sec61 inhibitors stabilize the plug domain in a closed state, thereby preventing the protein-translocation pore from opening. Our study provides the atomic details of Sec61-inhibitor interactions and the structural framework for further pharmacological studies and drug design.
•Efficient secretory production of proteins by Corynebacterium glutamicum.•An impressive variety of heterologous proteins has successfully been secreted.•Strategies for strain improvement and ...bioprocess optimization have been developed.
The Gram-positive soil bacterium Corynebacterium glutamicum has a long tradition in industry as a potent cell factory for the production of various amino acids. Besides this, in the last few years it became increasingly clear that this microorganism can also efficiently be used as a host organism for the expression and secretion of biotechnologically or pharmaceutically relevant heterologous proteins. In this review, first a short overview is given on the two main protein export pathways (Sec and Tat) of C. glutamicum that can be exploited for the transport of heterologous target proteins across the cytoplasmic membrane. Subsequently, the current knowledge on the successful use of C. glutamicum for the secretory production of an already impressive variety of heterologous proteins derived from different pro- and eukaryotic sources is summarized, whereby a special emphasis is given on the various optimization strategies and tools that have recently been developed and that now can be used to establish and further improve C. glutamicum as a secretory expression platform for the production of any desired heterologous target protein.
The twin-arginine protein translocation (Tat) system has been characterized in bacteria, archaea and the chloroplast thylakoidal membrane. This system is distinct from other protein transport systems ...with respect to two key features. Firstly, it accepts cargo proteins with an N-terminal signal peptide that carries the canonical twin-arginine motif, which is essential for transport. Second, the Tat system only accepts and translocates fully folded cargo proteins across the respective membrane. Here, we review the core essential features of folded protein transport via the bacterial Tat system, using the three-component TatABC system of
Escherichia coli
and the two-component TatAC systems of
Bacillus subtilis
as the main examples. In particular, we address features of twin-arginine signal peptides, the essential Tat components and how they assemble into different complexes, mechanistic features and energetics of Tat-dependent protein translocation, cytoplasmic chaperoning of Tat cargo proteins, and the remarkable proofreading capabilities of the Tat system. In doing so, we present the current state of our understanding of Tat-dependent protein translocation across biological membranes, which may serve as a lead for future investigations.
Unbalanced translocations can cause developmental delay (DD), intellectual disability (ID), growth problems, dysmorphic features, and congenital anomalies. They may arise de novo or may be inherited ...from a parent carrying a balanced rearrangement. It is estimated that 1/500 people is a balanced translocation carrier. The outcomes of different chromosomal rearrangements have the potential to reveal the functional consequences of partial trisomy or partial monosomy and can help guide genetic counseling for balanced carriers, and other young patients diagnosed with similar imbalances.
We performed clinical phenotyping and cytogenetic analyses of two siblings with a history of developmental delay (DD), intellectual disability (ID) and dysmorphic features.
The proband, a 38-year-old female, has a history of short stature, dysmorphic features and aortic coarctation. She underwent chromosomal microarray analysis, which identified partial monosomy of 4q and partial trisomy of 10p. Her brother, a 37-year-old male, has a history of more severe DD, behavioral problems, dysmorphic features, and congenital anomalies. Subsequently, karyotype confirmed two different unbalanced translocations in the siblings: 46,XX,der(4)t(4;10)(q33;p15.1) and 46,XY,der(10)t(4;10)(q33;p15.1), respectively. These chromosomal rearrangements represent two possible outcomes from a parent who is a carrier for a balanced translocation 46,XX,t(4;10)(q33;p15.1).
To our knowledge, this 4q and 10p translocation has not been described in literature. In this report we compare clinical features due to the composite effects of partial monosomy 4q with partial trisomy 10p and partial trisomy 4q with partial monosomy 10p. These findings speak to the relevance of old and new genomic testing, the viability of these segregation outcomes, and need for genetic counseling.
Self-complementing split fluorescent proteins (FPs) have been widely used for protein labeling, visualization of subcellular protein localization, and detection of cell-cell contact. To expand this ...toolset, we have developed a screening strategy for the direct engineering of self-complementing split FPs. Via this strategy, we have generated a yellow-green split-mNeonGreen2
that improves the ratio of complemented signal to the background of FP
-expressing cells compared to the commonly used split GFP
; as well as a 10-fold brighter red-colored split-sfCherry2
. Based on split sfCherry2, we have engineered a photoactivatable variant that enables single-molecule localization-based super-resolution microscopy. We have demonstrated dual-color endogenous protein tagging with sfCherry2
and GFP
, revealing that endoplasmic reticulum translocon complex Sec61B has reduced abundance in certain peripheral tubules. These new split FPs not only offer multiple colors for imaging interaction networks of endogenous proteins, but also hold the potential to provide orthogonal handles for biochemical isolation of native protein complexes.Split fluorescent proteins (FPs) have been widely used to visualise proteins in cells. Here the authors develop a screen for engineering new split FPs, and report a yellow-green split-mNeonGreen2 with reduced background, a red split-sfCherry2 for multicolour labeling, and its photoactivatable variant for super-resolution use.
Genetically engineered immune cells, like CAR T cell therapy and TCR T cell therapy, offer sustained cancer responses. Enabled by gene modification, these cells recognize and destroy tumors via ...synthetic receptors. The field addresses challenges such as low cell persistence, tumor microenvironment exhaustion, and off-tumor targeting risks. CRISPR-based editing simplifies gene deletion/insertion, overcoming CAR T therapy challenges. Examples include deleting negative regulators like PD-1 delays T cell exhaustion, and removing endogenous TCR reduces mispairing, mitigating toxicity. However, multiple gene edits done in batch pose chromosomal instability risks.
Using VECT mechanoporation, a non-viral biomechanical platform, we propose to deliver genome editing products targeting 2 genes into T cells sequentially to avoid generating multiple double stranded breaks at once. Compared to EP, VECT improves viability, recovery, and proliferation. With wild-type cas9, we knockout TCR and HLA in human T cells using VECT and electroporation. We determine repair time for double-strand breaks, quantifying gene editing efficiency, translocation rate, and memory markers.
Results show 6 hours between edits reduces translocation to 50%, and 24 hours eliminates it. VECT achieves 60% knockout for both targets, while EP achieves 80%. No editing rate reduction is observed in batch and sequential workflows up to 6 hours. No impact on proliferation or memory formation is seen with 6-hour sequential microfluidic compression, but 6-hour sequential electroporation causes delayed proliferation. For 24-hour sequential editing, both methods result in high cell death and growth delay. In future studies, batch and sequentially edited cells are transduced into CD19 CAR T cells for testing. In conclusion, VECT is superior for sequential multiplexed gene editing, reducing translocation risks.
Summary
The fine centromere structure in Robertsonian wheat‐rye translocation chromosomes exhibits variation among different translocation genotypes. Within extensively employed wheat‐rye 1RS.1BL ...translocation lines in wheat breeding, their translocated chromosomes frequently display fused centromere. Nevertheless, the mechanism governing the functionality of the fused centromere in 1RS.1BL translocated chromosomes remains to be clarified.
In this study, we investigated the fine centromere structure of the 1RS.1BL translocated chromosome through a combination of cytological and genomics methods. We found that only the rye‐derived centromere exhibits functional activity, whether in breeding applications or artificially synthesized translocation chromosomes.
The active rye‐derived centromere had higher proportion of young full‐length long terminal repeat retrotransposons (flLTR‐RTs) and more stable non‐B DNA structures, which may be beneficial toward transcription of centromeric repeats and CENH3 loading to maintain the activity of rye centromeres. High levels of DNA methylation and H3K9me2 were found in the inactive wheat‐derived centromeres, suggesting that it may play a crucial role in maintaining the inactive status of the wheat centromere.
Our works elucidate the fine structure of 1RS.1BL translocations and the potential mechanism of centromere inactivation in the fused centromere, contributing knowledge to the application of fused centromere in wheat breeding formation of new wheat‐rye translocation lines.
The essential process of protein secretion is achieved by the ubiquitous Sec machinery. In prokaryotes, the drive for translocation comes from ATP hydrolysis by the cytosolic motor-protein SecA, in ...concert with the proton motive force (PMF). However, the mechanism through which ATP hydrolysis by SecA is coupled to directional movement through SecYEG is unclear. Here, we combine all-atom molecular dynamics (MD) simulations with single molecule FRET and biochemical assays. We show that ATP binding by SecA causes opening of the SecY-channel at long range, while substrates at the SecY-channel entrance feed back to regulate nucleotide exchange by SecA. This two-way communication suggests a new, unifying 'Brownian ratchet' mechanism, whereby ATP binding and hydrolysis bias the direction of polypeptide diffusion. The model represents a solution to the problem of transporting inherently variable substrates such as polypeptides, and may underlie mechanisms of other motors that translocate proteins and nucleic acids.
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