A recA Null Mutation May Be Generated in Streptomyces coelicolor

ABSTRACT The recombinase RecA plays a crucial role in homologous recombination and the SOS response in bacteria. Although recA mutants usually are defective in homologous recombination and grow poorly, they nevertheless can be isolated in almost all bacteria. Previously, considerable difficulties were experienced by several laboratories in generating recA null mutations in Streptomyces, and the only recA null mutants isolated (from Streptomyces lividans) appeared to be accompanied by a suppressing mutation. Using gene replacement mediated by Escherichia coli-Streptomyces conjugation, we generated recA null mutations in a series of Streptomyces coelicolor A3(2) strains. These recA mutants were very sensitive to mitomycin C but only moderately sensitive to UV irradiation, and the UV survival curves showed wide shoulders, reflecting the presence of a recA-independent repair pathway. The mutants segregated minute colonies with low viability during growth and produced more anucleate spores than the wild type. Some crosses between pairs of recA null mutants generated no detectable recombinants, showing for the first time that conjugal recombination in S. coelicolor is recA mediated, but other mutants retained the ability to undergo recombination. The nature of this novel recombination activity is unknown.

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Deletion of an immunodominant Trypanosoma cruzi surface glycoprotein disrupts flagellum-cell adhesion

The Journal of Cell Biology ◽

10.1083/jcb.122.1.149 ◽

1993 ◽

Vol 122 (1) ◽

pp. 149-156 ◽

Cited By ~ 77

Author(s):

R Cooper ◽

AR de Jesus ◽

GA Cross

Keyword(s):

Trypanosoma Cruzi ◽

Gene Replacement ◽

Surface Glycoprotein ◽

Neomycin Phosphotransferase ◽

Flagellar Pocket ◽

Null Mutant ◽

Wild Type ◽

Western Blots ◽

Targeted Gene Replacement ◽

Null Mutants

Null mutants of the Trypanosoma cruzi insect stage-specific glycoprotein GP72 were created by targeted gene replacement. Targeting plasmids were constructed in which the neomycin phosphotransferase and hygromycin phosphotransferase genes were flanked by GP72 sequences. These plasmids were sequentially transfected into T. cruzi epimastigotes by electroporation. Southern blot analyzes indicated that precise replacement of the two genes had occurred. No aberrant rearrangements occurred at the GP72 locus and no GP72 gene sequences had been translocated elsewhere in the genome. Western blots confirmed that GP72 is not expressed in these null mutants. The morphology of the mutants is dramatically different from wild-type. In both mutant and wild-type parasites, the flagellum emerges from the flagellar pocket. In the null mutant the normal attachment of the flagellum to the cell membrane of the parasite is lost.

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Homologous Recombination Deficiency in Breast Cancer: A Clinical Review

JCO Precision Oncology ◽

10.1200/po.16.00031 ◽

2017 ◽

pp. 1-13 ◽

Cited By ~ 7

Author(s):

Wendie D. den Brok ◽

Kasmintan A. Schrader ◽

Sophie Sun ◽

Anna V. Tinker ◽

Eric Yang Zhao ◽

...

Keyword(s):

Breast Cancer ◽

Homologous Recombination ◽

Significant Proportion ◽

Clinical Use ◽

Breast Cancers ◽

Repair Pathway ◽

Wild Type ◽

Brca1 And Brca2 ◽

Homologous Recombination Deficiency ◽

Brca1 Brca2

BRCA1 and BRCA2 germline mutation–associated breast cancers are known to be deficient in the process of homologous recombination and often respond favorably to drugs targeting this important DNA repair pathway. There is emerging evidence that a significant proportion of patients with BRCA1/ BRCA2 wild-type breast cancer are also deficient in homologous recombination, and it is hypothesized that these patients may derive similar benefit from drugs targeting this pathway. Current research has focused on the development of a companion diagnostic to identify these sporadic BRCA-like tumors. This review outlines the various approaches that researchers have taken to predict homologous recombination deficiency as part of correlative biomarker work in various studies and clinical trials in breast cancer. As some of these tests of homologous recombination deficiency move closer to clinical use, understanding the approach and limitations of each is of relevance to clinicians who treat patients with breast cancer.

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Ability of a bacterial chromosome segment to invert is dictated by included material rather than flanking sequence.

Genetics ◽

10.1093/genetics/129.4.1021 ◽

1991 ◽

Vol 129 (4) ◽

pp. 1021-1032 ◽

Cited By ~ 4

Author(s):

M J Mahan ◽

J R Roth

Keyword(s):

Homologous Recombination ◽

Parent Strain ◽

Genetic Material ◽

Chromosomal Inversion ◽

Chromosome Segment ◽

Bacterial Chromosome ◽

Flanking Sequence ◽

Flanking Sequences ◽

Almost All

Abstract Homologous recombination between sequences present in inverse order within the same chromosome can result in inversion formation. We have previously shown that inverse order sequences at some sites (permissive) recombine to generate the expected inversion; no inversions are found when the same inverse order sequences flank other (nonpermissive) regions of the chromosome. In hopes of defining how permissive and nonpermissive intervals are determined, we have constructed a strain that carries a large chromosomal inversion. Using this inversion mutant as the parent strain, we have determined the "permissivity" of a series of chromosomal sites for secondary inversions. For the set of intervals tested, permissivity seems to be dictated by the nature of the genetic material present within the chromosomal interval being tested rather than the flanking sequences or orientation of this material in the chromosome. Almost all permissive intervals include the origin or terminus of replication. We suggest that the rules for recovery of inversions reflect mechanistic restrictions on the occurrence of inversions rather than lethal consequences of the completed rearrangement.

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The hobo Transposable Element Excises and Has Related Elements in Tephritid Species

Genetics ◽

10.1093/genetics/143.3.1339 ◽

1996 ◽

Vol 143 (3) ◽

pp. 1339-1347

Author(s):

Alfred M Handler ◽

Sheilachu P Gomez

Keyword(s):

Drosophila Melanogaster ◽

Ceratitis Capitata ◽

Bactrocera Dorsalis ◽

Parsimony Analysis ◽

Wild Type ◽

Anastrepha Suspensa ◽

Tephritid Species ◽

Mutant Strains ◽

Almost All ◽

Horizontal Transfers

Abstract Function of the Drosophila melanogaster hobo transposon in tephritid species was tested in transient embryonic excision assays. Wild-type and mutant strains of Anastrepha suspensa, Bactrocera dorsalis, B. cucurbitae, Ceratitis capitata, and Toxotrypana curvicauda all supported hobo excision or deletion both in the presence and absence of co-injected hobo transposase, indicating a permissive state for hobo mobility and the existence of endogenous systems capable of mobilizing hobo. In several strains hobo helper reduced excision. Excision depended on hobo sequences in the indicator plasmid, though almost all excisions were imprecise and the mobilizing systems appear mechanistically different from hobo. hobe-related sequences were identified in all species except T. curvicauda. Parsimony analysis yielded a subgroup including the B. cucurbitae and C. capitata sequences along with hobo and Hermes, and a separate, more divergent subgroup including the A. suspensa and B. dorsalis sequences. All of the sequences exist as multiple genomic elements, and a deleted form of the B. cucurbitae element exists in B. dorsalis. The hobo-related sequences are probably members of the hAT transposon family with some evolving from distant ancestor elements, while others may have originated from more recent horizontal transfers.

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Effects of B.1.1.7 and B.1.351 on COVID-19 Dynamics: A Campus Reopening Study

Archives of Computational Methods in Engineering ◽

10.1007/s11831-021-09638-y ◽

2021 ◽

Author(s):

Kevin Linka ◽

Mathias Peirlinck ◽

Amelie Schäfer ◽

Oguz Ziya Tikenogullari ◽

Alain Goriely ◽

...

Keyword(s):

Online Education ◽

The United States ◽

Wild Type ◽

Seir Model ◽

Population Mixing ◽

Santa Clara County ◽

Increased Risk ◽

Controversial Topic ◽

Almost All ◽

Local Reproduction

AbstractThe timing and sequence of safe campus reopening has remained the most controversial topic in higher education since the outbreak of the COVID-19 pandemic. By the end of March 2020, almost all colleges and universities in the United States had transitioned to an all online education and many institutions have not yet fully reopened to date. For a residential campus like Stanford University, the major challenge of reopening is to estimate the number of incoming infectious students at the first day of class. Here we learn the number of incoming infectious students using Bayesian inference and perform a series of retrospective and projective simulations to quantify the risk of campus reopening. We create a physics-based probabilistic model to infer the local reproduction dynamics for each state and adopt a network SEIR model to simulate the return of all undergraduates, broken down by their year of enrollment and state of origin. From these returning student populations, we predict the outbreak dynamics throughout the spring, summer, fall, and winter quarters using the inferred reproduction dynamics of Santa Clara County. We compare three different scenarios: the true outbreak dynamics under the wild-type SARS-CoV-2, and the hypothetical outbreak dynamics under the new COVID-19 variants B.1.1.7 and B.1.351 with 56% and 50% increased transmissibility. Our study reveals that even small changes in transmissibility can have an enormous impact on the overall case numbers. With no additional countermeasures, during the most affected quarter, the fall of 2020, there would have been 203 cases under baseline reproduction, compared to 4727 and 4256 cases for the B.1.1.7 and B.1.351 variants. Our results suggest that population mixing presents an increased risk for local outbreaks, especially with new and more infectious variants emerging across the globe. Tight outbreak control through mandatory quarantine and test-trace-isolate strategies will be critical in successfully managing these local outbreak dynamics.

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Engineered Phage Endolysin Eliminates Gardnerella Biofilm without Damaging Beneficial Bacteria in Bacterial Vaginosis Ex Vivo

Pathogens ◽

10.3390/pathogens10010054 ◽

2021 ◽

Vol 10 (1) ◽

pp. 54

Author(s):

Christine Landlinger ◽

Lenka Tisakova ◽

Vera Oberbauer ◽

Timo Schwebs ◽

Abbas Muhammad ◽

...

Keyword(s):

Bacterial Vaginosis ◽

Bactericidal Activity ◽

High Selectivity ◽

Ex Vivo ◽

Vaginal Microbiome ◽

Wild Type ◽

Wild Type Enzyme ◽

Promising Alternative ◽

First Time

Bacterial vaginosis is characterized by an imbalance of the vaginal microbiome and a characteristic biofilm formed on the vaginal epithelium, which is initiated and dominated by Gardnerella bacteria, and is frequently refractory to antibiotic treatment. We investigated endolysins of the type 1,4-beta-N-acetylmuramidase encoded on Gardnerella prophages as an alternative treatment. When recombinantly expressed, these proteins demonstrated strong bactericidal activity against four different Gardnerella species. By domain shuffling, we generated several engineered endolysins with 10-fold higher bactericidal activity than any wild-type enzyme. When tested against a panel of 20 Gardnerella strains, the most active endolysin, called PM-477, showed minimum inhibitory concentrations of 0.13–8 µg/mL. PM-477 had no effect on beneficial lactobacilli or other species of vaginal bacteria. Furthermore, the efficacy of PM-477 was tested by fluorescence in situ hybridization on vaginal samples of fifteen patients with either first time or recurring bacterial vaginosis. In thirteen cases, PM-477 killed the Gardnerella bacteria and physically dissolved the biofilms without affecting the remaining vaginal microbiome. The high selectivity and effectiveness in eliminating Gardnerella, both in cultures of isolated strains as well as in clinically derived samples of natural polymicrobial biofilms, makes PM-477 a promising alternative to antibiotics for the treatment of bacterial vaginosis, especially in patients with frequent recurrence.

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Deciphering the effects of kosmotrope and chaotrope salts during aqueous two phase extraction (ATPE) of polyphenolic compounds and glycoalkaloids from the leaves of a nutraceutical plant, Solanum retroflexum, with the aid of UPLC-QTOF-MS

Applied Biological Chemistry ◽

10.1186/s13765-021-00603-8 ◽

2021 ◽

Vol 64 (1) ◽

Author(s):

Tebogo Mokgehle ◽

Ntakadzeni Madala ◽

Wilson Gitari ◽

Nikita Tavengwa

Keyword(s):

Polar Compounds ◽

Ultra Performance Liquid Chromatography ◽

Polyphenolic Compounds ◽

Phase Extraction ◽

Two Phase ◽

Solanum Retroflexum ◽

Chaotropic Salts ◽

Almost All ◽

First Time ◽

Aqueous Two Phase Extraction

AbstractSolanum plants (Solanaceae) are renowned source of nutraceuticals and have widely been explored for their phytochemical constituents. This work investigated the effects of kosmotropic and chaotropic salts on the number of phytochemicals extracted from the leaves of a nutraceutical plant, Solanum retroflexum, and analyzed on the ultra-performance liquid chromatography hyphenated to a quadrupole time of flight mass spectrometer (UPLC-QTOF-MS) detector. Here, a total of 20 different compounds were putatively characterized. The majority of the identified compounds were polyphenols and glycoalkaloids. Another compound, caffeoyl malate was identified for the first time in this plant. Glycoalkaloids such as solanelagnin, solamargine, solasonine, β-solanine (I) and β-solanine (II) were found to be extracted by almost all the salts used herein. Kosmotrope salts, overall, were more efficient in extracting polar compounds with 4 more polyphenolic compounds extracted compared to the chaotropes. Chaotropes were generally more selective for the extraction of less polar compounds (glycoalkaloids) with 3 more extracted than the kosmotropes. The chaotrope and the kosmotrope that extracted the most metabolites were NaCl and Na2SO4, respectively, with 12 metabolites extracted for each salt. This work demonstrated that a comprehensive metabolome of S. retroflexum, more than what was previously reported on the same plant, can be achieved by application of kosmotropes and chaotropes as extractants with the aid of the Aqueous Two Phase Extraction approach. The best-performing salts, Na2SO4 or NaCl, could potentially be applied on a commercial scale, to meet the ever-growing demand of the studied metabolites. The Aqueous Two Phase Extraction technique was found to be efficient in simultaneous extraction of multiple metabolites which can be applied in metabolomics.

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Elimination of Introns at the Drosophila suppressor-of-forked Locus by P-Element-Mediated Gene Conversion Shows That an RNA Lacking a Stop Codon is Dispensable

Genetics ◽

10.1093/genetics/143.1.345 ◽

1996 ◽

Vol 143 (1) ◽

pp. 345-351

Author(s):

Carol J Williams ◽

Kevin O'Hare

Keyword(s):

X Chromosome ◽

Stop Codon ◽

Alternative Polyadenylation ◽

Gene Replacement ◽

P Element ◽

Wild Type ◽

Genomic Location ◽

Chromosomal Breaks ◽

White Locus ◽

Cleavage Stimulation Factor

Abstract The suppressor of forked [su(f)] locus affects the phenotype of mutations caused by transposable element insertions at unlinked loci. It encodes a putative 84-kD protein with homology to two proteins involved in mRNA 3′ end processing; the product of the yeast RNA14 gene and the 77-kD subunit of human cleavage stimulation factor. Three su(f) mRNAs are produced by alternative polyadenylation. The 2. 6 and 2.9-kb mRNAs encode the same 84-kD protein while a 1.3-kb RNA, which terminates within the fourth intron, is unusual in having no stop codon. Using P-element-mediated gene replacement we have copied sequences from a transformation construct into the su(f) gene creating a su(f) allele at the normal genomic location that lacks the first five introns. This allele is viable and appears wild type for su(f) function, demonstrating that the 1.3-kb RNA and the sequences contained within the deleted introns are dispensable for su(f) function. Compared with studies on gene replacement at the white locus, chromosomal breaks at su(f) appear to be less efficiently repaired from ectopic sites, perhaps because of the location of su(f) at the euchromatin/heterochromatin boundary on the X chromosome.

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Seed morphology uncovers 1500 years of vine agrobiodiversity before the advent of the Champagne wine

Scientific Reports ◽

10.1038/s41598-021-81787-3 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Vincent Bonhomme ◽

Jean-Frédéric Terral ◽

Véronique Zech-Matterne ◽

Sarah Ivorra ◽

Thierry Lacombe ◽

...

Keyword(s):

Middle Ages ◽

Climatic Changes ◽

Seed Morphology ◽

Crucial Aspect ◽

Wild Type ◽

Grape Seeds ◽

The Social ◽

Reference Collection ◽

Grape Varieties ◽

First Time

AbstractA crucial aspect of viticulture is finally unveiled as the historical dynamics of its agrobiodiversity are described in the Champagne region for the first time. Outline analyses were carried out to compare the morphology of archaeological grape seeds from Troyes and Reims (first c. AD to fifteenth c. AD) with that of a reference collection of modern seeds, including wild vines and traditional grape varieties, believed to be ancient and characteristic of the French vine heritage. This allows us to document the chronological dynamics of the use of the wild Vitis type and of the diversity of the varieties used, based on morphological disparity. After showing the existence of morphological types corresponding to geographical groups, we highlight a geochronological dynamic. Our results show that the wild type is used throughout the series, up to the Middle Ages. In addition, domestic forms, morphologically related to southern varietal groups, are very early involved in the Champagne grape agrodiversity. The groups corresponding to the typical grape varieties of today do not appear until the second millennium. These previously unsuspected dynamics are discussed in light of the social, societal and climatic changes documented for the period.

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Functional Assessment of 12 Rare Allelic CYP2C9 Variants Identified in a Population of 4773 Japanese Individuals

Journal of Personalized Medicine ◽

10.3390/jpm11020094 ◽

2021 ◽

Vol 11 (2) ◽

pp. 94

Author(s):

Masaki Kumondai ◽

Akio Ito ◽

Evelyn Marie Gutiérrez Rico ◽

Eiji Hishinuma ◽

Akiko Ueda ◽

...

Keyword(s):

Enzymatic Activity ◽

Three Dimensional ◽

Warfarin Dose ◽

Catalytic Efficiency ◽

Therapeutic Window ◽

Wild Type ◽

Drug Metabolizing Enzyme ◽

Novel Variants ◽

Metabolizing Enzyme ◽

Almost All

Cytochrome P450 2C9 (CYP2C9) is an important drug-metabolizing enzyme that contributes to the metabolism of approximately 15% of clinically used drugs, including warfarin, which is known for its narrow therapeutic window. Interindividual differences in CYP2C9 enzymatic activity caused by CYP2C9 genetic polymorphisms lead to inconsistent treatment responses in patients. Thus, in this study, we characterized the functional differences in CYP2C9 wild-type (CYP2C9.1), CYP2C9.2, CYP2C9.3, and 12 rare novel variants identified in 4773 Japanese individuals. These CYP2C9 variants were heterologously expressed in 293FT cells, and the kinetic parameters (Km, kcat, Vmax, catalytic efficiency, and CLint) of (S)-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation were estimated. From this analysis, almost all novel CYP2C9 variants showed significantly reduced or null enzymatic activity compared with that of the CYP2C9 wild-type. A strong correlation was found in catalytic efficiencies between (S)-warfarin 7-hydroxylation and tolbutamide 4-hydroxylation among all studied CYP2C9 variants. The causes of the observed perturbation in enzyme activity were evaluated by three-dimensional structural modeling. Our findings could clarify a part of discrepancies among genotype–phenotype associations based on the novel CYP2C9 rare allelic variants and could, therefore, improve personalized medicine, including the selection of the appropriate warfarin dose.

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