scholarly journals Characterization of ori and parS-like functions in secondary genome replicons in Deinococcus radiodurans

2020 ◽  
Vol 4 (1) ◽  
pp. e202000856
Author(s):  
Ganesh K Maurya ◽  
Hari S Misra
Keyword(s):  
Functional Significance ◽  
Copy Number ◽  
Deinococcus Radiodurans ◽  
The Other ◽  
Origin Of Replication ◽  
Wild Type ◽  
Genome Maintenance ◽  
Fluorescent Reporter ◽  
Operator System

The mechanisms underlying multipartite genome maintenance and its functional significance in extraordinary radioresistance of Deinococcus radiodurans are not well understood. The sequences upstream to parAB operons in chrII (cisII) and MP (cisMP) could stabilize an otherwise, non-replicative colE1 plasmid, in D. radiodurans. DnaA and cognate ParB proteins bound specifically with cisII and cisMP elements. The ΔcisII and ΔcisMP cells showed the reduced copy number of cognate replicons and radioresistance as compared with wild type. Fluorescent reporter–operator system inserted in chrI, chrII, and MP in wild type and cisII mutants showed the presence of all three replicons in wild-type cells. Although chrI was present in all the ΔcisII and ΔcisMP cells, nearly half of these cells had chrII and MP, respectively, and the other half had the reduced number of foci representing these replications. These results suggested that cisII and cisMP elements contain both origin of replication and parS-like functions and the secondary genome replicons (chrII and MP) are maintained independent of chrI and have roles in radioresistance of D. radiodurans.

scholarly journals Characterization of an Unstable Allele of the Arabidopsis HY4 Locus

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1575-1585
Author(s):  
Edward P Bruggemann ◽  
Bernard Doan ◽  
Korie Handwerger ◽  
Gisela Storz
Keyword(s):  
Fast Neutron ◽  
Blue Light ◽  
Large Deletion ◽  
The Other ◽  
Epigenetic Mechanisms ◽  
Loss Of Function ◽  
Wild Type ◽  
Unusual Behavior ◽  
Recessive Lethal

Abstract The Arabidopsis HY4 gene encodes the nonessential blue light photoreceptor CRY1. Loss-of-function hy4 mutants have an elongated hypocotyl phenotype after germination under blue light. We previously analyzed 20 independent hy4 alleles produced by fast neutron mutagenesis. These alleles were grouped into two classes based on their genetic behavior and corresponding deletion size: (1) null hy4 alleles that were semidominant over wild type and contained small or moderate-sized deletions at HY4 and (2) null hy4 alleles that were recessive lethal and contained large HY4 deletions. Here we describe one additional fast neutron hy4 mutant, B144, that did not fall into either of these two classes. Mutant B144 was isolated as a heterozygote with an intermediate hy4 phenotype. One allele from this mutant, hy4-B144Δ, contains a large deletion at HY4 and is recessive lethal. The other allele from this mutant, HY4-B144*, appears to be intact and functional but is unstable and spontaneously converts to a nonfunctional hy4 allele. In addition, HY4-B144* is lethal in homozygotes and suppresses local recombination. We discuss genetic and epigenetic mechanisms that may account for the unusual behavior of the HY4-B144* allele.

scholarly journals Overlapping oriC and centromere-like functions in secondary genome replicons determine their maintenance independent of chromosome I in Deinococcus radiodurans

2020 ◽  
Author(s):  
Ganesh K Maurya ◽  
Hari S. Misra
Keyword(s):  
Radiation Resistance ◽  
Copy Number ◽  
Deinococcus Radiodurans ◽  
Specific Interactions ◽  
Wild Type ◽  
Γ Radiation ◽  
E Coli ◽  
Copy Numbers ◽  
Type Pattern ◽  
Chromosome I

AbstractThe Deinococcus radiodurans multipartite genome system (MGS) consists of chromosome I (ChrI) and secondary genome elements; Chr II and megaplasmid (MP). The sequences upstream to parAB operons in Chr II (cisII) and MP (cisMP) helped an E. coli plasmid maintenance in D. radiodurans and showed sequence specific interactions with DnaA and ParBs. The cells devoid of cisII (ΔcisII) or cisMP (ΔcisMP) showed reduced γ radiation resistance and copy number of Chr II and MP. Fluorescent Reporter-Operator System (FROS) developed for ChrI, ChrII and MP in ΔcisII or ΔcisMP mutants showed no change in wild type pattern of Chr I localization. However, the relative copy numbers of Chr II and MP had reduced while anucleate cells had increased in mutants. These results suggested that cisII and cisMP elements contain both ori and centromere-like functions, and like other MGS bacteria, the Chr I and secondary genome are maintained independently in D. radiodurans.

scholarly journals Discovery and Characterization of Native Deinococcus radiodurans Promoters for Tunable Gene Expression

2019 ◽  
Vol 85 (21) ◽  
Author(s):  
Angela Chen ◽  
Mark W. Sherman ◽  
Cynthia Chu ◽  
Natalia Gonzalez ◽  
Tulshi Patel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Metabolic Engineering ◽  
Protein Production ◽  
Deinococcus Radiodurans ◽  
Promoter Sequence ◽  
Current Standard ◽  
Fluorescent Reporter ◽  
Content Type ◽  
Oxidative Stresses

ABSTRACT The potential utilization of extremophiles as a robust chassis for metabolic engineering applications has prompted interest in the use of Deinococcus radiodurans for bioremediation efforts, but current applications are limited by the lack of availability of genetic tools, such as promoters. In this study, we used a combined computational and experimental approach to identify and screen 30 predicted promoters for expression in D. radiodurans using a fluorescent reporter assay. The top eight candidates were further characterized, compared to currently available promoters, and optimized for engineering through minimization for use in D. radiodurans. Of these top eight, two promoter regions, PDR_1261 and PrpmB, were stronger and more consistent than the most widely used promoter sequence in D. radiodurans, PgroES. Furthermore, half of the top eight promoters could be minimized by at least 20% (to obtain final sequences that are approximately 24 to 177 bp), and several of the putative promoters either showed activity in Escherichia coli or were D. radiodurans specific, broadening the use of the promoters for various applications. Overall, this work introduces a suite of novel, well-characterized promoters for protein production and metabolic engineering in D. radiodurans. IMPORTANCE The tolerance of the extremophile, Deinococcus radiodurans, to numerous oxidative stresses makes it ideal for bioremediation applications, but many of the tools necessary for metabolic engineering are lacking in this organism compared to model bacteria. Although native and engineered promoters have been used to drive gene expression for protein production in D. radiodurans, very few have been well characterized. Informed by bioinformatics, this study expands the repertoire of well-characterized promoters for D. radiodurans via thorough characterization of eight putative promoters with various strengths. These results will help facilitate tunable gene expression, since these promoters demonstrate strong and consistent performance compared to the current standard, PgroES. This study also provides a methodology for high-throughput promoter identification and characterization using fluorescence in D. radiodurans. The promoters identified in this study will facilitate metabolic engineering of D. radiodurans and enable its use in biotechnological applications ranging from bioremediation to synthesis of commodity chemicals.

scholarly journals Construction and Characterization of a Derivative of Neisseria gonorrhoeae Strain MS11 Devoid of AllopaGenes

2012 ◽  
Vol 194 (23) ◽  
pp. 6468-6478 ◽  
Author(s):  
Adriana LeVan ◽  
Lindsey I. Zimmerman ◽  
Amanda C. Mahle ◽  
Karen V. Swanson ◽  
Philip DeShong ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
The Other ◽  
Flow Cytometry Analysis ◽  
Wild Type ◽  
Homogeneous Population ◽  
Host Cell Interactions ◽  
Strain Ms11 ◽  
Adherence Ability

ABSTRACTTo better understand the role of Opa in gonococcal infections, we created and characterized a derivative of MS11 (MS11Δopa) that had the coding sequence for all 11 Opa proteins deleted. The MS11Δopa bacterium lost the ability to bind to purified lipooligosaccharide (LOS). While nonpiliated MS11Δopa and nonpiliated Opa-expressing MS11 cells grew at the same rate, nonpiliated MS11Δopa cells rarely formed clumps of more than four bacteria when grown in broth with vigorous shaking. Using flow cytometry analysis, we demonstrated that MS11Δopa produced a homogeneous population of bacteria that failed to bind monoclonal antibody (MAb) 4B12, a MAb specific for Opa. Opa-expressing MS11 cells consisted of two predominant populations, where ∼85% bound MAb 4B12 to a significant level and the other population bound little if any MAb. Approximately 90% of bacteria isolated from a phenotypically Opa-negative colony (a colony that does not refract light) failed to bind MAb 4B12; the remaining 10% bound MAb to various degrees. Piliated MS11Δopa cells formed dispersed microcolonies on ME180 cells which were visually distinct from those of piliated Opa-expressing MS11 cells. When Opa expression was reintroduced into MS11Δopa, the adherence ability of the strain recovered to wild-type levels. These data indicate that Opa contributes to both bacterium-bacterium and bacterium-host cell interactions.

scholarly journals Role of π Dimers in Coupling (“Handcuffing”) of Plasmid R6K's γ ori Iterons

2005 ◽  
Vol 187 (11) ◽  
pp. 3779-3785 ◽  
Author(s):  
Selvi Kunnimalaiyaan ◽  
Ross B. Inman ◽  
Sheryl A. Rakowski ◽  
Marcin Filutowicz
Keyword(s):  
Copy Number ◽  
Origin Of Replication ◽  
Wild Type ◽  
Rep Protein ◽  
Copy Number Control ◽  
Proposed Model ◽  
Rep Proteins ◽  
Origin Function ◽  
Replication Initiator

ABSTRACT One proposed mechanism of replication inhibition in iteron-containing plasmids (ICPs) is “handcuffing,” in which the coupling of origins via iteron-bound replication initiator (Rep) protein turns off origin function. In minimal R6K replicons, copy number control requires the interaction of plasmid-encoded π protein with the seven 22-bp iterons of the γ origin of replication. Like other related Rep proteins, π exists as both monomers and dimers. However, the ability of π dimers to bind iterons distinguishes R6K from most other ICPs, where only monomers have been observed to bind iterons. Here, we describe experiments to determine if monomers or dimers of π protein are involved in the formation of handcuffed complexes. Standard ligation enhancement assays were done using π variants with different propensities to bind iterons as monomers or dimers. Consistent with observations from several ICPs, a hyperreplicative variant (π·P106L∧F107S) exhibits deficiencies in handcuffing. Additionally, a novel dimer-biased variant of π protein (π·M36A∧M38A), which lacks initiator function, handcuffs iteron-containing DNA more efficiently than does wild-type π. The data suggest that π dimers mediate handcuffing, supporting our previously proposed model of handcuffing in the γ ori system. Thus, dimers of π appear to possess three distinct inhibitory functions with respect to R6K replication: transcriptional autorepression of π expression, in cis competition (for origin binding) with monomeric activator π, and handcuffing-mediated inhibition of replication in trans.

scholarly journals A rare human centenarian variant of SIRT6 enhances genome stability and interaction with Lamin A

2021 ◽  
Author(s):  
Vera Gorbunova ◽  
Matthew Simon ◽  
Greg Trombline ◽  
Jiping Yang ◽  
Eric J Earley ◽  
...  
Keyword(s):  
Genome Stability ◽  
Strand Break ◽  
Human Longevity ◽  
Lamin A ◽  
Ashkenazi Jewish ◽  
Wild Type ◽  
Genome Maintenance ◽  
Dna Double Strand Break ◽  
Cellular Pathways

Sirtuin 6 (SIRT6) is a deacylase and mono-ADP ribosyl transferase (mADPr) enzyme involved in multiple cellular pathways implicated in the regulation of aging and metabolism. Targeted sequencing identified a SIRT6 allele containing two linked substitutions (N308K/A313S) as enriched in Ashkenazi Jewish (AJ) centenarians as compared to AJ control individuals. Characterization of this SIRT6 (centSIRT6) allele demonstrated it to be a stronger suppressor of LINE1 retrotransposons, confer enhanced stimulation of DNA double strand break repair, and more robust cancer cell killing compared to the wild type. Surprisingly, centSIRT6 displayed weaker deacetylase activity, but stronger mADPr activity, over a range of NAD+ concentrations and substrates. Additionally, centSIRT6 displayed a stronger interaction with Lamin A/C (LMNA), which correlated with enhanced ribosylation of LMNA. Our results suggest that enhanced SIRT6 function contributes to human longevity by improving genome maintenance via increased mADPr activity and enhanced interaction with LMNA.

Ultrastructural study of human fetal appendix: A “novel” cytoplasmic organelle

1996 ◽  
Vol 54 ◽  
pp. 930-931
Author(s):  
K. M. Siddiqui
Keyword(s):  
Functional Significance ◽  
Lymphoid Tissue ◽  
Vermiform Appendix ◽  
Cell Types ◽  
The Other ◽  
Cytoplasmic Organelle ◽  
Similar Function ◽  
Chronic Appendicitis ◽  
Close Observation

Vermiform appendix (VA) present in humans and other vertebrates may have a similar function to gut-associated lymphoepithelial tissue (GALT). The function of GALT in the gastrointestinal tract (GIT) is well documented. The functional significance of VA, as a “lymphoid” tissue in developing human fetus has not been established. However, VA is the narrowest part of GIT that protrudes from the base of cecum at the ileocecal juncture. Several diseases such as chronic appendicitis, mucocele, argentaffin carcinoma, and developmental diverticula causing inflammation are associated with VA (Delikaris, et al., 1983). An agenesis of VA and complications associated with it in a child born to a mother who had taken thalidomide during her pregnancy has been documented (Bremner & Mooney, 1978). These pathologies and also occurance of appendicitis with cystic fibrosis (Bockman, 1983) suggest a functional significance, and warrants a close observation of VA in human fetuses. On the other hand, studies detailing the subcellular architecture and characterization of various cell types of it have been limited,

scholarly journals Characterization of the ccpA Gene ofEnterococcus faecalis: Identification of Starvation-Inducible Proteins Regulated by CcpA

2000 ◽  
Vol 182 (20) ◽  
pp. 5799-5806 ◽  
Author(s):  
Céline Leboeuf ◽  
Laurence Leblanc ◽  
Yanick Auffray ◽  
Axel Hartke
Keyword(s):  
Deinococcus Radiodurans ◽  
Pleiotropic Effect ◽  
Amino Acid Sequences ◽  
Wild Type ◽  
Phosphotransferase System ◽  
Homologous Proteins ◽  
Mutant Strains ◽  
Terminal Amino ◽  
Mutant Cells

ABSTRACT Inactivation of ccpA in Enterococcus faecalis leads to reduction of the growth rate, derepression of the galKETR operon in the presence of a mixture of glucose and galactose, and reduction of transcription of ldh in the presence of glucose. Moreover, the E. faecalis ccpA gene fully complements a Bacillus subtilis ccpA mutant, arguing for similar functions of these two homologous proteins. Protein comparison on two-dimensional gels from the wild-type cells and theccpA mutant cells revealed a pleiotropic effect of the mutation on gene expression. The HPr protein of the carbohydrate-phosphotransferase system was identified by microsequencing, and a modification of its phosphorylation state was observed between the wild-type and the mutant strains. Moreover, at least 16 polypeptides are overexpressed in the mutant, and 6 are repressed. Interestingly, 13 of the 16 polypeptides whose synthesis is enhanced in the mutant were also identified as glucose starvation proteins. The N-terminal amino acid sequences of four of them match sequences deduced from genes coding for l-serine dehydratase, dihydroxyacetone kinase (two genes), and a protein of unknown function from Deinococcus radiodurans.

A very rapid in situ Kikuchi technique to determine relative crystal misorientation

1988 ◽  
Vol 46 ◽  
pp. 830-831
Author(s):  
J. I. Bennetch
Keyword(s):  
Electron Beam ◽  
Analytical Techniques ◽  
Superplastic Forming ◽  
The Other ◽  
Kikuchi Pattern ◽  
Kikuchi Line ◽  
Line Analysis

In a recent study of the superplastic forming (SPF) behavior of certain Al-Li-X alloys, the relative misorientation between adjacent (sub)grains proved to be an important parameter. It is well established that the most accurate way to determine misorientation across boundaries is by Kikuchi line analysis. However, the SPF study required the characterization of a large number of (sub)grains in each sample to be statistically meaningful, a very time-consuming task even for comparatively rapid Kikuchi analytical techniques.In order to circumvent this problem, an alternate, even more rapid in-situ Kikuchi technique was devised, eliminating the need for the developing of negatives and any subsequent measurements on photographic plates. All that is required is a double tilt low backlash goniometer capable of tilting ± 45° in one axis and ± 30° in the other axis. The procedure is as follows. While viewing the microscope screen, one merely tilts the specimen until a standard recognizable reference Kikuchi pattern is centered, making sure, at the same time, that the focused electron beam remains on the (sub)grain in question.

Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

1982 ◽  
Vol 47 (03) ◽  
pp. 197-202 ◽  
Author(s):  
Kurt Huber ◽  
Johannes Kirchheimer ◽  
Bernd R Binder
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Gel Filtration ◽  
Chain Structure ◽  
The Other ◽  
Single Chain ◽  
Apparent Homogeneity ◽  
Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

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