Biology subjects

Gutierrez, C.

Publications and source records attributed to Gutierrez, C..

At least 19 recordsLinked to original sources

bioRxiv2026

AADAT-Driven Metabolic Control of Malate and CoQ10 Shapes Immune Evasion in Triple-Negative Breast Cancer

Compared to other subtypes of breast cancer, triple-negative breast cancers (TNBC) have fewer treatment options and exhibit a worse prognosis. Through integrated transcriptomic, metabolomic, immunohistochemical, spatial, and clinical analyses, we identify the mitochondrial enzyme, -aminoadipate aminotransferase (AADAT) as a previously unrecognized metabolic immune checkpoint in TNBC. AADAT mRNA and protein were significantly upregulated in human TNBC, and high AADAT expression was associated with reduced intra-tumoral CD8 T-cell density and inferior survival. Genetic silencing of AADAT in orthotopic murine TNBC models curtailed primary tumor growth and distant metastasis in a CD8 T-cell-dependent manner, enhanced effector T-cell activation, and sensitized tumors to dual PD-1/CTLA-4 blockade. Mechanistically, unbiased metabolomics showed increased malate levels after AADAT knockdown. Additionally, 4-hydroxyphenylpyruvate, an essential precursor for coenzyme Q10(CoQ10) biosynthesis, decreased following AADAT knockdown, suggesting an impaired mitochondrial electron transport chain. CoQ10 supplementation restored metabolic balance and reversed malate accumulation caused by AADAT knockdown, indicating that AADAT helps maintain CoQ10-supported redox homeostasis, thereby preventing malate buildup and export. Notably, malate addition directly boosted CD8 T-cell oxidative metabolism, increased the NAD/NADH ratio and reactive oxygen species, and augmented TNF- and IFN-{gamma} production. In vivo, malate supplementation in drinking water phenocopied AADAT knockdown, restored the response to paclitaxel plus anti-PD-1 therapy in multiple independent syngeneic TNBC models with de novo or acquired resistance to immunotherapy, reduced tumor burden, and prolonged survival. In patient cohorts, higher spatially clustered intra-tumoral malate is associated with co-localization of functional CD8 T cells, decreased exhausted T-cell neighborhoods, and superior post-chemotherapy outcomes. These data position AADAT as a central metabolic orchestrator of immune escape in TNBC and nominate oral malate as a readily translatable adjuvant to reverse chemo-immunotherapy resistance in TNBC. Statement of SignificanceAADAT defines a metabolic-immune axis driving immune evasion and therapy resistance in triple-negative breast cancer. Blocking AADAT or administering oral malate reactivates CD8 T-cell immunity and sensitizes chemo-immunotherapy-resistant tumors to these agents. These findings uncover a readily translatable metabolic vulnerability with potential to improve outcomes for patients with aggressive breast cancer subtypes.

cancer biology↗

bioRxiv2025

Rat somatic genome editing enables ER+ breast cancer modeling

Genetically engineered mouse models have advanced cancer research but often fail to capture key features of certain human tumors. Rats, with distinct physiology and tumor biology, offer a powerful alternative, yet their use has been constrained by technical barriers to genome editing. Here, we report efficient somatic genome editing in rats, enabling both Indel and substitution mutations. We then apply this approach to model estrogen receptor (ER)-positive breast cancer, which accounts for [~]70% of human cases but remains poorly represented in mice. The resulting rat tumors reproduce hallmarks of human ER+ breast cancer, including ductal histology, hormone responsiveness, and immune-microenvironmental features. By contrast, identical genetic alterations in mice failed to yield ER+ tumors, underscoring critical species differences in tumorigenesis. Together, this work establishes a versatile platform for rapid generation of clinically relevant rat tumor models, opening new avenues to dissect tumor biology, therapeutic response, and immune interactions in previously inaccessible cancer subtypes.

cancer biology↗

bioRxiv2025

The role of FRUITFULL controlling cell cycle during early flower development revealed by time series snRNA-seq experiments.

BACKGROUNDStarting from pools of undifferentiated cells, plants generate new organs post-embryonically in response to external and endogenous signals. This requires a dynamic coordination of cell division with cellular growth and differentiation regulatory programs. However, little is known how this coordination is achieved at the molecular level during flower development. RESULTSWe used time-series single-nucleus RNA sequencing (snRNA-seq) experiments of synchronized Arabidopsis thaliana flower developmental stages to characterize the transcriptome dynamics and the connections between cell cycle and developmental regulatory programs during early flower development. The results show a bifurcation between transcriptional trajectories corresponding to cell cycle progression and floral development. We identify the regulation of the cell cycle inhibitor KIP-RELATED PROTEIN 2 (KRP2) by FRUITFULL (FUL) as a key regulatory point on this bifurcation point, and validate the importance of this regulation in vivo. CONCLUSIONSOur work illustrates how time-series snRNA-seq experiments can be used to identify bifurcation points between regulatory programs and to identify candidate regulators on these bifurcations. In particular, we identify the regulation of KRP2 by FUL as an important regulatory point to balance cell division and developmental differentiation in plants.

plant biology↗

bioRxiv2025

Loss of the Y chromosome drives cancer metabolic reprogramming

Metabolic reprogramming promotes cancer aggressiveness and an immune-suppressive tumor microenvironment. Loss of the Y chromosome (LOY) drives both phenotypes in bladder cancer (BC). We investigated the hypothesis that LOY leads to metabolic reprogramming using untargeted metabolomic profiling of human BC cells and analysis of pan-cancer transcriptomic datasets. This revealed that aerobic glycolysis is activated in LOY BC cells. Since prior work showed that expression of collagen receptor DDR2 drives BC progression and DDR2 is a regulator of tumor metabolism, we investigated if DDR2 is implicated in metabolic reprogramming of LOY-tumors. Analysis of scRNAseq data from 251 patients with 12 tumor types found that LOY and DDR2 expression promote aerobic glycolysis, and this was confirmed by metabolomics. Deletion of DDR2 in LOY BC cells reduced glycolytic flux, inhibited cell proliferation, reduced EMT and stemness features, and promoted apoptosis. Our data provide a rationale for using LOY as a tumor selection biomarker for DDR2 targeted therapeutics. TeaserLoss of the Y chromosome augments glycolytic metabolism in bladder cancer cells, and this is in part dependent on the collagen receptor DDR2.

cancer biology↗

bioRxiv2025

PacL-organized membrane-associated effluxosomes coordinate multi-metal resistance in Mycobacterium tuberculosis

Metal ion homeostasis is crucial for bacterial pathogens to withstand metal-induced stress during infection. However, the mechanisms underlying bacterial resistance to metal stress remain incompletely understood, particularly how bacteria coordinate responses to simultaneous exposure to multiple metals. Here, we uncover a previously unrecognized mechanism by which Mycobacterium tuberculosis, the causative agent of tuberculosis, orchestrates a coordinated response to multi-metal stress. We demonstrate that M. tuberculosis assembles dynamic, membrane-associated platforms, organized by PacL proteins, that confer resistance to multiple metals simultaneously. PacL proteins function as scaffolds, clustering multiple P-type ATPase (P-ATPase) pumps, CtpC, CtpG, and CtpV, into functional complexes we term "effluxosomes". Our findings show that PacL proteins are critical for stabilizing CtpG within membrane-associated clusters, conferring cadmium tolerance, while CtpC serves as a backup, promoting cross-resistance to both zinc and cadmium. Using super-resolution microscopy and single-particle tracking, we elucidate the 3D structure and dynamics of effluxosomes in the mycobacterial membrane. We further demonstrate that conserved residues within the transmembrane domain of PacL proteins are crucial for the assembly of dynamic effluxosomes, which are essential for P-ATPase activity. Additionally, we reveal that PacL1 exhibits metallochaperone activity, binding zinc, cadmium, and copper via a conserved C-terminal motif. Proximity labeling further identifies an extensive PacL1 interaction network, encompassing multiple proteins involved in stress adaptation. Our findings introduce effluxosomes as dynamic, membrane-associated efflux machineries that mediate coordinated multi-metal resistance in M. tuberculosis, providing new insights into bacterial metal homeostasis and unveiling potential antimicrobial targets.

microbiology↗

bioRxiv2025

GsMTx-4 Reduces Mechanosensitivity in a Model of Schwannomatosis-related Pain

Patients with schwannomatosis (SWN) develop multiple tumors along major peripheral nerves, with most experiencing significant pain, though each patients symptoms are unique. Neuropathic, nociceptive, and inflammatory pain types have been reported, but many patients describe severe pain when a schwannoma is palpated or even lightly touched. Currently, the only effective treatment for pain relief is surgical removal. We are investigating the root causes of tumor-induced pain. In some cases, tumor growth increases pressure on nearby nerves, resulting in pain. Additionally, schwannoma cells in culture secrete proinflammatory cytokines into the surrounding medium. This conditioned medium (CM) sensitizes sensory neurons to painful stimuli both in vitro and in vivo. When injected into the glabrous skin of a mouse hindpaw, CM from painful schwannomas increases neuron sensitivity to light touch, as demonstrated by a fourfold reduction in paw withdrawal threshold (measured using the Von Frey assay) one hour post-injection (p = 0.006), with effects persisting for 24 hours (p = 0.002).We hypothesize that this increase in sensitivity is linked to mechanosensitive ion channels (MSCs), which detect pressure and stretch. These channels can be blocked by the peptide GsMTx-4. This peptide penetrates deeper into cell membranes under mechanical pressure to block MSCs from opening without affecting other ion channels. When co-injected with CM into the mouse hindpaw, 10 {micro}M GsMTx-4 prevents heightened sensitivity to light touch. Moreover, GsMTx-4 can reverse hyperalgesia, restoring withdrawal thresholds to baseline levels. Thus, local injection of GsMTx-4 near painful tumors presents a promising, minimally invasive therapeutic approach for SWN patients. SignificancePain is a confounding comorbidity in the multiple tumor syndrome schwannomatosis. Patients harbor benign peripheral nerve sheath tumors that rarely become malignant or cause neurological deficits. Yet, patients undergo numerous surgeries for the removal of painful tumors. A non-invasive treatment for tumor-related pain is in dire need. We are examining the small peptide GsMTx-4, a blocker of mechanosensitive ion channels, as a potential therapy for painful tumors in the context of schwannomatosis.

neuroscience↗

bioRxiv2025

Undergraduate GPA does not predict success in PhD programs for cohorts of MS students at two minority-serving institutions

Master of Science (MS) research training programs funded by organizations such as the NIH, the NSF as well as private corporations represent the potential for significant interventions for student success, especially for marginalized or underrepresented and first-generation students, to bridge the gap between undergraduate studies and doctoral programs. These students often face serious challenges during their undergraduate years, such as navigating unfamiliar academic systems, balancing demanding coursework with work responsibilities, and fulfilling family obligations. These and other systemic pressures can impede their academic progress and opportunities for research experience. We therefore asked how their undergraduate GPA impacted success in the PhD in terms of (i) acceptance, (ii) completion, and (iii) time to degree after participating in a funded MS research training program. We examined data collected at San Francisco State University and California State University, Los Angeles over a 30-year period because they (i) had similar student demographics, (ii) were institutions with strong MS degrees, and (iii) had an infrastructure (established offices) to coordinate various training programs. We found that high GPA did not predict greater success in entering or completing the PhD, or in time to degree. This study, therefore, demonstrates that undergraduates from diverse groups with lower undergraduate GPA levels can benefit from a structured MS research training program in reaching their goal of becoming a PhD scientist. Moreover, it indicates that more holistic approaches in admissions are required, including limiting the use of a GPA below 3.0 as an early filter to eliminate applicants. These MS programs appear to play a crucial role as a bridge to PhD studies and are instrumental in enhancing diversity within STEM fields. We suggest that a strategy for success is to provide infrastructure in the form of a coordinated office housing the training grants as a means of structuring mentorship and professional development for under-represented and first generation students.

scientific communication and education↗

bioRxiv2024

Warm temperature modifies cell fates to reduce stomata production in Arabidopsis

O_LIStomatal abundance decrease in Arabidopsis triggered by warm-temperature is attributed to PIF4-mediated repression of SPEECHLESS (SPCH) expression. We identified the unknown developmental and transcriptional basis of this adaptive response. C_LIO_LIWe traced stomatal lineages in vivo using cell-identity marker lines and mutants, quantified epidermal traits and conducted RNA sequencing under oscillating temperatures. C_LIO_LIProlonged warm-temperature or PIF4-overexpression altered cell-fates, inducing diverted stomatal precursors (DPs) that lacked stomatal fate, accounting for stomata reduction. DPs originated from meristemoids that lost SPCH expression, lacked MUTE expression and exited the cell cycle. Short warm-temperature pulses allowed later recovery of SPCH expression, and did not induce DPs or stomata reduction. Comparison of transcriptomes obtained during warm-temperature pulses with stomatal lineage cell-specific profiles identified gene expression changes and contrasted their reversibility. Though warm-temperature silenced key stomatal drivers, most lineages formed stomata through partly modified transcriptional landscapes that promoted uncommitted cell identities and included alternative pathways. C_LIO_LIExpression changes in stomatal regulators and cell-fate changes explain lineage progression under fluctuating temperatures. Since short-term temperature oscillations prevail in natural conditions, the requirement of long warm-temperature exposure to trigger DPs would prevent stomata reduction by occasional temperature rises. Promoting uncommitted lineage stages provides flexibility to stomatal development under environmental changes. C_LI

plant biology↗

bioRxiv2024

The topography of DNA replication origins in Eukarya: GGN clusters, landmark nucleosomes, CDC6 and G4 structures

BackgroundWe recently identified tandem repeats of GGN triplets as a sequence motif strongly enriched in the DNA replication origins (ORIs) of the Arabidopsis thaliana genome, where it correlates with the ORI strength quantified through short nascent strands sequencing. ResultsHere we show that clusters of four or more GGN repeats separated by short tracts (<7 nucleotides) are present in more than 65% ORIs of six model eukaryotic organisms: Leishmania major, Arabidopsis thaliana, Caenorhabditis elegans, Drosophila melanogaster, Mus musculus and Homo sapiens. The measured percentages vary with the experimental technique adopted for ORI determination. For all studied techniques and organisms, ORIs are significantly prone to be located within or near strong GGN clusters, although these are neither necessary nor sufficient for ORI activation. Interestingly, for same experimental technique, the association between ORIs and GGN clusters was stronger al later developmental stages for all organisms for which we could perform this comparison. In accordance with a biophysical model of nucleosome positioning, the GGN clusters strongly favor nucleosome occupancy. At the same time, both GGN clusters and ORIs occur frequently within 1kb from nucleosome-depleted regions (NDR). We propose a structural model based on chromatin secondary structure in which the NDR and the well-positioned nucleosome at the GGN are close in space, which may favor functional interactions. We hypothesize that the presence of GGN at ORIs arose in part for promoting the above nucleosome organization Moreover, at least in Arabidopsis, the replication protein CDC6 is very strongly associated with GGN clusters. NMR experiments showed that clusters of at least four GGN can form G-quadruplex (G4) in vitro. Our data support the view that GGN clusters are formed through the interplay of mutational processes (GC skew at ORIs plus triplet expansion), and that similar mutational processes, i.e. AT skew at transcription start sites (TSS) and ORIs, might facilitate the formation of the NDR, thus favoring the evolvability of chromatin. ConclusionsGGN clusters are easily evolvable sequence motifs enriched at ORIs of eukaryotic genomes. They favor G4 secondary structure and a nucleosome organization that may unify the apparent discrepancy between ORIs of higher eukaryotes and yeast.

genomics↗

bioRxiv2023

Nuclear GSH import precedes coordinated cell cycle changes during regeneration

The plasticity of plant cells underlies their wide capacity to regenerate, with increasing evidence in plants and animals implicating cell cycle dynamics in cellular reprogramming. To investigate the cell cycle during cellular reprogramming, we developed a comprehensive set of cell cycle phase markers in the Arabidopsis root. Using single-cell RNA-seq profiles and live imaging during regeneration, we found that a subset of cells near an ablation injury dramatically increases division rate by truncating G1. Cells in G1 undergo a transient nuclear peak of glutathione (GSH) prior to coordinated entry into S phase followed by rapid divisions and cellular reprogramming. A symplastic block of the ground tissue impairs regeneration, which is rescued by exogenous GSH. We propose a model in which GSH from the outer tissues is released upon injury licensing an exit from G1 near the wound to induce rapid cell division and reprogramming.

plant biology↗

bioRxiv2023

Maintenance of cell wall remodeling and vesicle production are connected in Mycobacterium tuberculosis

Pathogenic and nonpathogenic mycobacteria secrete extracellular vesicles (EVs) under various conditions. EVs produced by Mycobacterium tuberculosis (Mtb) have raised significant interest for their potential in cell communication, nutrient acquisition, and immune evasion. However, the relevance of vesicle secretion during tuberculosis infection remains unknown due to the limited understanding of mycobacterial vesicle biogenesis. We have previously shown that a transposon mutant in the LCP-related gene virR (virRmut) manifested a strong attenuated phenotype during experimental macrophage and murine infections, concomitant to enhanced vesicle release. In this study, we aimed to understand the role of VirR in the vesicle production process in Mtb. We employ genetic, transcriptional, proteomics, ultrastructural and biochemical methods to investigate the underlying processes explaining the enhanced vesiculogenesis phenomenon observed in the virRmut. Our results establish that VirR is critical to sustain proper cell permeability via regulation of cell envelope remodeling possibly through the interaction with similar cell envelope proteins, which control the link between peptidoglycan and arabinogalactan. These findings advance our understanding of mycobacterial extracellular vesicle biogenesis and suggest that these set of proteins could be attractive targets for therapeutic intervention.

microbiology↗

bioRxiv2023

Cell-cycle status of male and female gametes during Arabidopsis reproduction

Fertilization in Arabidopsis thaliana is a highly coordinated process that begins with a pollen tube delivering the two sperm cells into the embryo sac. Each sperm cell can then fertilize either the egg or the central cell to initiate embryo or endosperm development, respectively. The success of this double fertilization process requires a tight cell cycle synchrony between the male and female gametes to allow karyogamy (nuclei fusion). However, the cell cycle status of the male and female gametes during fertilization still remains elusive as DNA quantification and DNA replication assays have given conflicting results1-4. Here, to reconcile these results, we quantified the DNA replication state by DNA sequencing and performed microscopic analyses of fluorescent markers covering all the phases of the cell cycle. We show that male and female gametes in Arabidopsis are both arrested prior to DNA replication at maturity and initiate their DNA replication only during fertilization.

plant biology↗

bioRxiv2022

The Arabidopsis SHORTROOT network coordinates shoot apical meristem development with auxin dependent lateral organ initiation

Unlike animals, plants have the capacity to produce new organs post-embryonically throughout their entire life cycle. This is due to stem cells present in the shoot and the root apical meristems (SAM and RAM, respectively). In the SAM, stem cells are located in the central zone (CZ) where they divide slowly. Stem cell daughters are displaced laterally and enter the peripheral zone (PZ). Here, their mitotic activity increases, and lateral organ primordia (LOP) are formed. How the spatial arrangement of these different domains is initiated and controlled during SAM growth and development, and how sites of LOP are determined in the PZ is not yet completely understood. In the RAM, the GRAS family transcription factor SHORTROOT (SHR) acts as a master regulator of signalling pathways that maintain the root stem cell niche and control formation of ground tissue layers. We hypothesized that SHR could perform a similar role in the SAM, and found that SHR, together with its target transcription factors SCARECROW (SCR), SCARECROW-LIKE23 (SCL23) and JACKDAW (JKD), controls shoot meristem size by regulating cell division rates, and promotes formation of lateral organs. SHR, SCR, SCL23 and JKD are expressed in very distinct patterns in the SAM. Where these expression domains overlap, they can physically interact to activate expression of the key cell cycle regulator CYCLIND6;1 (CYCD6;1) and thereby promote the formation of new cell layers. In the PZ, upregulation of SHR expression at sites of organ initiation depends on the phytohormone auxin, acting through the auxin response factor MONOPTEROS (MP) and auxin response elements in the SHR promoter. In the CZ, the SHR-target SCL23 physically interacts with WUS, a key regulator of stem cell maintenance, and both SCL23 and WUS expression are subject to negative feedback regulation from stem cells through the CLAVATA signalling pathway. Together, our findings illustrate how SHR-dependent transcription factor complexes act in different domains of the shoot meristem to mediate cell division and auxin dependent organ initiation in the PZ, and coordinate this activity with stem cell maintenance in the CZ of the SAM.

plant biology↗

bioRxiv2022

Stem cell regulators control a G1 duration gradient in the plant root meristem

In meristems, where new plant organs initiate, key stem cell regulators have been identified, but their link to cell cycle progression remains unclear. Here, we show that the root meristem has a positional gradient of G1 duration that ranges from [~]2 h near the meristem boundary to more than 20 h in stem cells and early derivatives. Mutants in the PLETHORA (PLT) genes shortened G1 length and flattened its gradient. Computer modeling of an incoherent feed-forward loop (IFFL) predicted the inference of a negative regulatory pathway. We propose that PLT genes play opposing roles, maintaining meristem and stem cell activity and inhibiting G1 progression through the CDK inhibitor KRP5, a PLT target, and RBR1. This establishes a previously undescribed proximal-distal feature of the root meristem in which a G1 duration gradient is shaped by stem cell and meristem maintenance regulators.

plant biology↗

bioRxiv2021

Mycobacterial resistance to zinc poisoning requires assembly of P-ATPase-containing membrane metal efflux platforms

Transition metals are toxic at high concentrations. The P1B-ATPase metal exporter CtpC/Rv3270 is required for resistance to zinc poisoning in the human pathogen Mycobacterium tuberculosis. Here, we discovered that zinc resistance also depends on the chaperone-like protein PacL1/Rv3269. PacL1 bound Zn2+, but unlike PacL1 and CtpC, the PacL1 metal-binding motif (MBM) was required only at high zinc concentrations. PacL1 co-localized with CtpC in dynamic microdomains within the mycobacterial plasma membrane. Microdomain formation did not require flotillins nor the PacL1 MBM. Instead, loss of the PacL1 Glutamine/Alanine repeats led to loss of CtpC and sensitivity to zinc. PacL1 and CtpC are within the same operon, and homologous PacL1-P1B-ATPase pairs are widely distributed within and across prokaryotes. PacL1 colocalized and functioned redundantly with PacL orthologs in Mycobacterium tuberculosis. Overall, our study suggests that PacL proteins are scaffolds that assemble P-ATPase-containing metal efflux platforms, a novel type of functional membrane microdomain that underlies bacterial resistance to metal poisoning.

microbiology↗

bioRxiv2020

The Polycomb group protein MEDEA controls cell proliferation and embryonic patterning in Arabidopsis

Establishing the body plan of a multicellular organism relies on precisely orchestrated cell divisions coupled with pattern formation. In animals, cell proliferation and embryonic patterning are regulated by Polycomb group (PcG) proteins that form various multisubunit complexes (Grossniklaus and Paro, 2014). The evolutionary conserved Polycomb Repressive Complex 2 (PRC2) trimethylates histone H3 at lysine 27 (H3K27me3) and comes in different flavors in the model plant Arabidopsis thaliana (Forderer et al., 2016; Grossniklaus and Paro, 2014). The histone methyltransferase MEDEA (MEA) is part of the FERTILIZATION INDEPENDENT SEED (FIS)-PRC2 required for seed development4. Although embryos derived from mea mutant egg cells show morphological abnormalities (Grossniklaus et al., 1998), defects in the development of the placenta-like endosperm are considered the main cause of seed abortion (Kinoshita et al., 1999; Scott et al., 1998), and a role of FIS-PRC2 in embryonic patterning was dismissed (Bouyer et al., 2011; Leroy et al., 2007). Here, we demonstrate that endosperm lacking MEA activity sustains normal embryo development and that embryos derived from mea mutant eggs abort even in presence of a wild-type endosperm because MEA is required for embryonic patterning and cell lineage determination. We show that, similar to PcG proteins in mammals, MEA regulates embryonic growth by repressing the transcription of core cell cycle components. Our work demonstrates that Arabidopsis embryogenesis is under epigenetic control of maternally expressed PcG proteins, revealing that PRC2 was independently recruited to control embryonic cell proliferation and patterning in animals and plants.

developmental biology↗

bioRxiv2019

FBL17 targets CDT1a for degradation in early S-phase to prevent Arabidopsis genome instability

Maintenance of genome integrity depends on controlling the availability of DNA replication initiation proteins, e.g., CDT1, a component of the pre-replication complexes that regulates chromatin licensing for replication. To understand the evolutionary history of CDT1 regulation, we have identified the mechanisms involved in CDT1 dynamics. During cell cycle, CDT1a starts to be loaded early after mitotic exit and maintains high levels until the G1/S transition. Soon after the S-phase onset, CDT1a is rapidly degraded in a proteasome-dependent manner. Plant cells use a specific SCF-mediated pathway that relies on the FBL17 F-box protein for CDT1a degradation, which is independent of CUL4a-containing complexes. A similar oscillatory pattern occurs in endoreplicating cells, where CDT1a is loaded just after finishing the S-phase. CDT1a is necessary to maintain genome stability, an ancient strategy although unique proteins and mechanisms have evolved in different eukaryotic lineages to ensure its degradation during S-phase.\n\nImpact statementThe DNA replication protein CDT1a is crucial for genome integrity and is targeted for proteasome degradation just after S-phase initiation by FBL17 in proliferating and endoreplicating cells of Arabidopsis

plant biology↗

bioRxiv2019

An improved Xer-cise technology for the generation of multiple unmarked mutants in Mycobacteria

Xer-cise is a technique using antibiotic resistance cassettes flanked by dif sites allowing spontaneous and accurate excision from bacterial chromosomes with a high frequency through the action of the cellular recombinase XerCD. Here, we report a significant improvement of Xer-cise in Mycobacteria. Zeocin-resistance cassettes flanked by variants of the natural Mycobacterium tuberculosis dif site were constructed and shown to be effective tools to construct multiple unmarked mutations in M. tuberculosis and in the model species Mycobacterium smegmatis. The dif site variants harbor mutations in the central region and can therefore not recombine with the wild type or other variants, resulting in mutants of increased genetic stability. The herein described method should be generalizable to virtually any transformable bacterial species.\n\nMethod summarydif-ZeoR-dif cassettes are used to replace non-essential genes in mycobacterial genome through recombineering. Spontaneous excision of the cassette is carried out under the action of the recombinase XerCD, resulting in unmarked deletions. Subsequent rounds of mutagenesis using cassettes flanked by a range of dif site variants allow construction of multiple mutants in which the different dif sites cannot recombine which each other, yielding stable genetic constructs.

microbiology↗