International Journal of Molecular Sciences

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976 KiB

Open AccessArticle

A Chimeric UDP-Glucose Pyrophosphorylase Produced by Protein Engineering Exhibits Sensitivity to Allosteric Regulators

by Matías D. Asención Diez, Ana C. Ebrecht, Lucila I. Martínez, Mabel C. Aleanzi, Sergio A. Guerrero, Miguel A. Ballícora and Alberto A. Iglesias

Int. J. Mol. Sci. 2013, 14(5), 9703-9721; https://doi.org/10.3390/ijms14059703 - 6 May 2013

Cited by 7 | Viewed by 7532

Abstract

In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc) or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase) or unregulated UDP-Glc PPase (EC 2.7.7.9). In this work, we characterized the UDP-Glc PPase [...] Read more.

In bacteria, glycogen or oligosaccharide accumulation involves glucose-1-phosphate partitioning into either ADP-glucose (ADP-Glc) or UDP-Glc. Their respective synthesis is catalyzed by allosterically regulated ADP-Glc pyrophosphorylase (EC 2.7.7.27, ADP-Glc PPase) or unregulated UDP-Glc PPase (EC 2.7.7.9). In this work, we characterized the UDP-Glc PPase from Streptococcus mutans. In addition, we constructed a chimeric protein by cutting the C-terminal domain of the ADP-Glc PPase from Escherichia coli and pasting it to the entire S. mutans UDP-Glc PPase. Both proteins were fully active as UDP-Glc PPases and their kinetic parameters were measured. The chimeric enzyme had a slightly higher affinity for substrates than the native S. mutans UDP-Glc PPase, but the maximal activity was four times lower. Interestingly, the chimeric protein was sensitive to regulation by pyruvate, 3-phosphoglyceric acid and fructose-1,6-bis-phosphate, which are known to be effectors of ADP-Glc PPases from different sources. The three compounds activated the chimeric enzyme up to three-fold, and increased the affinity for substrates. This chimeric protein is the first reported UDP-Glc PPase with allosteric regulatory properties. In addition, this is a pioneer work dealing with a chimeric enzyme constructed as a hybrid of two pyrophosphorylases with different specificity toward nucleoside-diphospho-glucose and our results turn to be relevant for a deeper understanding of the evolution of allosterism in this family of enzymes. Full article

(This article belongs to the Special Issue Molecular Cut and Paste)

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433 KiB

Open AccessArticle

Off-Target Effect of Endogenous siRNA Derived from RMRP in Human Cells

by Yoshiko Maida, Satoru Kyo, Timo Lassmann, Yoshihide Hayashizaki and Kenkichi Masutomi

Int. J. Mol. Sci. 2013, 14(5), 9305-9318; https://doi.org/10.3390/ijms14059305 - 29 Apr 2013

Cited by 11 | Viewed by 7368

Abstract

Endogenous siRNAs (endo-siRNAs) are key regulators of RNA silencing in plants and worms; however, the biogenesis and function of endogenous siRNAs in mammals remain largely unknown. We previously demonstrated that human telomerase reverse transcriptase produces a self-targeting endogenous siRNA from non-coding RMRP RNA [...] Read more.

Endogenous siRNAs (endo-siRNAs) are key regulators of RNA silencing in plants and worms; however, the biogenesis and function of endogenous siRNAs in mammals remain largely unknown. We previously demonstrated that human telomerase reverse transcriptase produces a self-targeting endogenous siRNA from non-coding RMRP RNA via RNA-dependent RNA polymerase (RdRP) activity. Here, we investigated whether the endo-siRNA derived from RMRP targets other genes in addition to RMRP. Four algorithms for microRNA target prediction were used to identify possible targets of the endo-siRNA, and the phytanoyl-CoA hydroxylase-interacting protein-like gene (PHYHIPL) was identified as the most promising candidate. The 3' UTR of PHYHIPL was found to contain three possible target sites with perfect seed pairing; deletion of each of these sites resulted in recovery of upstream luciferase expression. In addition, sequence-specific inhibition of the RMRP-derived endo-siRNA increased expression of PHYHIPL mRNA. The results described here suggest that the endo-siRNA uses silencing mechanisms that are similar to those used by microRNAs for gene silencing. To our knowledge, this study is the first confirmation of the off-target effect of human endogenous siRNA produced by RdRP activity. Full article

(This article belongs to the Special Issue Molecular Cut and Paste)

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2137 KiB

Open AccessArticle

Thermostable Mismatch-Recognizing Protein MutS Suppresses Nonspecific Amplification during Polymerase Chain Reaction (PCR)

by Kenji Fukui, Yoshitaka Bessho, Atsuhiro Shimada, Shigeyuki Yokoyama and Seiki Kuramitsu

Int. J. Mol. Sci. 2013, 14(3), 6436-6453; https://doi.org/10.3390/ijms14036436 - 21 Mar 2013

Cited by 11 | Viewed by 10470

Abstract

Polymerase chain reaction (PCR)-related technologies are hampered mainly by two types of error: nonspecific amplification and DNA polymerase-generated mutations. Here, we report that both errors can be suppressed by the addition of a DNA mismatch-recognizing protein, MutS, from a thermophilic bacterium. Although it [...] Read more.

Polymerase chain reaction (PCR)-related technologies are hampered mainly by two types of error: nonspecific amplification and DNA polymerase-generated mutations. Here, we report that both errors can be suppressed by the addition of a DNA mismatch-recognizing protein, MutS, from a thermophilic bacterium. Although it had been expected that MutS has a potential to suppress polymerase-generated mutations, we unexpectedly found that it also reduced nonspecific amplification. On the basis of this finding, we propose that MutS binds a mismatched primer-template complex, thereby preventing the approach of DNA polymerase to the 3' end of the primer. Our simple methodology improves the efficiency and accuracy of DNA amplification and should therefore benefit various PCR-based applications, ranging from basic biological research to applied medical science. Full article

(This article belongs to the Special Issue Molecular Cut and Paste)

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1433 KiB

Open AccessArticle

Quick, Selective and Reversible Photocrosslinking Reaction between 5-Methylcytosine and 3-Cyanovinylcarbazole in DNA Double Strand

by Kenzo Fujimo, Kaoru Konishi-Hiratsuka and Takashi Sakamoto

Int. J. Mol. Sci. 2013, 14(3), 5765-5774; https://doi.org/10.3390/ijms14035765 - 12 Mar 2013

Cited by 14 | Viewed by 7084

Abstract

Selective photocrosslinking reaction between 3-cyanovinylcarbazole nucleoside (^CNVK) and 5-methylcytosine (^mC), which is known as epigenetic modification in genomic DNA, was developed. The reaction was completely finished within 5 s of 366 nm irradiation, and the rate of this photocrosslinking [...] Read more.

Selective photocrosslinking reaction between 3-cyanovinylcarbazole nucleoside (^CNVK) and 5-methylcytosine (^mC), which is known as epigenetic modification in genomic DNA, was developed. The reaction was completely finished within 5 s of 366 nm irradiation, and the rate of this photocrosslinking reaction was ca. 30-fold higher than that in the case of unmodified normal cytosine. There were no significant differences in the thermodynamic parameters and the kinetics of hybrid formation of oligonucleotide (ODN) containing ^CNVK and its complementary ODN containing C or ^mC at the photocrosslinking site, and suggesting that the quick and selective photoreaction has potential for the selective detection of ^mC in the DNA strand via the photocrosslinking reaction. Full article

(This article belongs to the Special Issue Molecular Cut and Paste)

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243 KiB

Open AccessArticle

Molecular Diversity Assessment Using Sequence Related Amplified Polymorphism (SRAP) Markers in Vicia faba L.

by Salem S. Alghamdi, Sulieman A. Al-Faifi, Hussein M. Migdadi, Muhammad Altaf Khan, Ehab H. EL-Harty and Megahed H. Ammar

Int. J. Mol. Sci. 2012, 13(12), 16457-16471; https://doi.org/10.3390/ijms131216457 - 4 Dec 2012

Cited by 53 | Viewed by 8674

Abstract

Sequence-related amplified polymorphism (SRAP) markers were used to assess the genetic diversity and relationship among 58 faba bean (Vicia faba L.) genotypes. Fourteen SRAP primer combinations amplified a total of 1036 differently sized well-resolved peaks (fragments), of which all were polymorphic with [...] Read more.

Sequence-related amplified polymorphism (SRAP) markers were used to assess the genetic diversity and relationship among 58 faba bean (Vicia faba L.) genotypes. Fourteen SRAP primer combinations amplified a total of 1036 differently sized well-resolved peaks (fragments), of which all were polymorphic with a 0.96 PIC value and discriminated all of the 58 faba bean genotypes. An average pairwise similarity of 21% was revealed among the genotypes ranging from 2% to 65%. At a similarity of 28%, UPGMA clustered the genotypes into three main groups comprising 78% of the genotypes. The local landraces and most of the Egyptian genotypes in addition to the Sudan genotypes were grouped in the first main cluster. The advanced breeding lines were scattered in the second and third main clusters with breeding lines from the ICARDA and genotypes introduced from Egypt. At a similarity of 47%, all the genotypes formed separated clusters with the exceptions of Hassawi 1 and Hassawi 2. Group analysis of the genotypes according to their geographic origin and type showed that the landraces were grouped according to their origin, while others were grouped according to their seed type. To our knowledge, this is the first application of SRAP markers for the assessment of genetic diversity in faba bean. Such information will be useful to determine optimal breeding strategies to allow continued progress in faba bean breeding. Full article

(This article belongs to the Special Issue Molecular Cut and Paste)

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Review

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536 KiB

Open AccessReview

Bacterial Cellular Engineering by Genome Editing and Gene Silencing

by Nobutaka Nakashima and Kentaro Miyazaki

Int. J. Mol. Sci. 2014, 15(2), 2773-2793; https://doi.org/10.3390/ijms15022773 - 18 Feb 2014

Cited by 42 | Viewed by 18284

Abstract

Genome editing is an important technology for bacterial cellular engineering, which is commonly conducted by homologous recombination-based procedures, including gene knockout (disruption), knock-in (insertion), and allelic exchange. In addition, some new recombination-independent approaches have emerged that utilize catalytic RNAs, artificial nucleases, nucleic acid [...] Read more.

Genome editing is an important technology for bacterial cellular engineering, which is commonly conducted by homologous recombination-based procedures, including gene knockout (disruption), knock-in (insertion), and allelic exchange. In addition, some new recombination-independent approaches have emerged that utilize catalytic RNAs, artificial nucleases, nucleic acid analogs, and peptide nucleic acids. Apart from these methods, which directly modify the genomic structure, an alternative approach is to conditionally modify the gene expression profile at the posttranscriptional level without altering the genomes. This is performed by expressing antisense RNAs to knock down (silence) target mRNAs in vivo. This review describes the features and recent advances on methods used in genomic engineering and silencing technologies that are advantageously used for bacterial cellular engineering. Full article

(This article belongs to the Special Issue Molecular Cut and Paste)

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632 KiB

Open AccessReview

The Enzyme-Mediated Direct Reversal of a Dithymine Photoproduct in Germinating Endospores

by Linlin Yang and Lei Li

Int. J. Mol. Sci. 2013, 14(7), 13137-13153; https://doi.org/10.3390/ijms140713137 - 25 Jun 2013

Cited by 9 | Viewed by 8173

Abstract

Spore photoproduct lyase (SPL) repairs a special thymine dimer, 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct, or SP, in germinating endospores. SP is the exclusive DNA photo-damaging product found in endospores; its generation and swift repair by SPL are responsible for the spores’ [...] Read more.

Spore photoproduct lyase (SPL) repairs a special thymine dimer, 5-thyminyl-5,6-dihydrothymine, which is commonly called spore photoproduct, or SP, in germinating endospores. SP is the exclusive DNA photo-damaging product found in endospores; its generation and swift repair by SPL are responsible for the spores’ extremely high UV resistance. Early in vivo studies suggested that SPL utilizes a direct reversal strategy to repair SP in the absence of light. Recently, it has been established that SPL belongs to the radical S-adenosylmethionine (SAM) superfamily. The enzymes in this superfamily utilize a tri-cysteine CXXXCXXC motif to bind a [4Fe-4S] cluster. The cluster provides an electron to the S-adenosylmethionine (SAM) to reductively cleave its C5'-S bond, generating a reactive 5'-deoxyadenosyl (5'-dA) radical. This 5'-dA radical abstracts the proR hydrogen atom from the C6 carbon of SP to initiate the repair process; the resulting SP radical subsequently fragments to generate a putative thymine methyl radical, which accepts a back-donated H atom to yield the repaired TpT. The H atom donor is suggested to be a conserved cysteine141 in B. subtilis SPL; the resulting thiyl radical likely interacts with a neighboring tyrosine99 before oxidizing the 5'-dA to 5'-dA radical and, subsequently, regenerating SAM. These findings suggest SPL to be the first enzyme in the large radical SAM superfamily (>44,000 members) to utilize a radical transfer pathway for catalysis; its study should shed light on the mechanistic understanding of the SAM regeneration process in other members of the superfamily. Full article

(This article belongs to the Special Issue Molecular Cut and Paste)

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296 KiB

Open AccessReview

Tailoring the Models of Transcription

by Alena Pance

Int. J. Mol. Sci. 2013, 14(4), 7583-7597; https://doi.org/10.3390/ijms14047583 - 8 Apr 2013

Cited by 3 | Viewed by 7074

Abstract

Molecular biology is a rapidly evolving field that has led to the development of increasingly sophisticated technologies to improve our capacity to study cellular processes in much finer detail. Transcription is the first step in protein expression and the major point of regulation [...] Read more.

Molecular biology is a rapidly evolving field that has led to the development of increasingly sophisticated technologies to improve our capacity to study cellular processes in much finer detail. Transcription is the first step in protein expression and the major point of regulation of the components that determine the characteristics, fate and functions of cells. The study of transcriptional regulation has been greatly facilitated by the development of reporter genes and transcription factor expression vectors, which have become versatile tools for manipulating promoters, as well as transcription factors in order to examine their function. The understanding of promoter complexity and transcription factor structure offers an insight into the mechanisms of transcriptional control and their impact on cell behaviour. This review focuses on some of the many applications of molecular cut-and-paste tools for the manipulation of promoters and transcription factors leading to the understanding of crucial aspects of transcriptional regulation. Full article

(This article belongs to the Special Issue Molecular Cut and Paste)

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Graphical abstract

868 KiB

Open AccessReview

Cut-and-Paste of DNA Using an Artificial Restriction DNA Cutter

by Makoto Komiyama

Int. J. Mol. Sci. 2013, 14(2), 3343-3357; https://doi.org/10.3390/ijms14023343 - 5 Feb 2013

Cited by 11 | Viewed by 8236

Abstract

DNA manipulations using a completely chemistry-based DNA cutter (ARCUT) have been reviewed. This cutter, recently developed by the authors, is composed of Ce(IV)/EDTA complex and two strands of pseudo-complementary peptide nucleic acid. The site-selective scission proceeds via hydrolysis of targeted phosphodiester linkages, so [...] Read more.

DNA manipulations using a completely chemistry-based DNA cutter (ARCUT) have been reviewed. This cutter, recently developed by the authors, is composed of Ce(IV)/EDTA complex and two strands of pseudo-complementary peptide nucleic acid. The site-selective scission proceeds via hydrolysis of targeted phosphodiester linkages, so that the resultant scission fragments can be easily ligated with other fragments by using DNA ligase. Importantly, scission-site and site-specificity of the cutter are freely tuned in terms of the Watson–Crick rule. Thus, when one should like to manipulate DNA according to the need, he or she does not have to think about (1) whether appropriate “restriction enzyme sites” exist near the manipulation site and (2) whether the site-specificity of the restriction enzymes, if any, are sufficient to cut only the aimed position without chopping the DNA at non-targeted sites. Even the human genome can be manipulated, since ARCUT can cut the genome at only one predetermined site. Furthermore, the cutter is useful to promote homologous recombination in human cells, converting a site to desired sequence. The ARCUT-based DNA manipulation should be promising for versatile applications. Full article

(This article belongs to the Special Issue Molecular Cut and Paste)

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