scholarly journals Systematically investigating the key features of the nuclease deactivated Cpf1 for tunable multiplex genetic regulation

2018 ◽  
Author(s):  
Chensi Miao ◽  
Huiwei Zhao ◽  
Long Qian ◽  
Chunbo Lou
Keyword(s):  
Gene Regulation ◽  
Computational Algorithm ◽  
Dynamic Range ◽  
Genetic Regulation ◽  
Direct Repeat ◽  
Repeat Sequence ◽  
Gene Repression ◽  
Efficient Design ◽  
Francisella Novicida ◽  
Cas9 Protein

AbstractWith a unique crRNA processing capability, the CRISPR associated Cpf1 protein holds great potential for multiplex gene regulation. Unlike the well-studied Cas9 protein, however, conversion of Cpf1 to a transcription regulator and its related properties have not been systematically explored yet. In this study, we investigated the mutation schemes and crRNA requirements for the nuclease deactivated Cpf1 (dCpf1). By shortening the direct repeat sequence, we obtained genetically stable crRNA co-transcripts and improved gene repression with multiplex targeting. A screen of diversity-enriched PAM library was designed to investigate the PAM-dependency of gene regulation by dCpf1 from Francisella novicida and Lachnospiraceae bacterium. We found novel PAM patterns that elicited strong or medium gene repressions. Using a computational algorithm, we predicted regulatory outputs for all possible PAM sequences, which spanned a large dynamic range that could be leveraged for regulatory purposes. These newly identified features will facilitate the efficient design of CRISPR-dCpf1 based systems for tunable multiplex gene regulation.

scholarly journals Novel Gene Regulation in Normal and Abnormal Spermatogenesis

Cells ◽  
2021 ◽  
Vol 10 (3) ◽  
pp. 666
Author(s):  
Li Du ◽  
Wei Chen ◽  
Zixin Cheng ◽  
Si Wu ◽  
Jian He ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Leydig Cells ◽  
Molecular Mechanisms ◽  
New Technologies ◽  
Genetic Regulation ◽  
Myoid Cells ◽  
Epigenetic Factors ◽  
Future Directions ◽  
New Genes ◽  
Human Spermatogenesis

Spermatogenesis is a complex and dynamic process which is precisely controlledby genetic and epigenetic factors. With the development of new technologies (e.g., single-cell RNA sequencing), increasingly more regulatory genes related to spermatogenesis have been identified. In this review, we address the roles and mechanisms of novel genes in regulating the normal and abnormal spermatogenesis. Specifically, we discussed the functions and signaling pathways of key new genes in mediating the proliferation, differentiation, and apoptosis of rodent and human spermatogonial stem cells (SSCs), as well as in controlling the meiosis of spermatocytes and other germ cells. Additionally, we summarized the gene regulation in the abnormal testicular microenvironment or the niche by Sertoli cells, peritubular myoid cells, and Leydig cells. Finally, we pointed out the future directions for investigating the molecular mechanisms underlying human spermatogenesis. This review could offer novel insights into genetic regulation in the normal and abnormal spermatogenesis, and it provides new molecular targets for gene therapy of male infertility.

scholarly journals Isolation and Characterization of a 1.7-kb Transposable Element from a Mutator Line of Maize

Genetics ◽  
1987 ◽  
Vol 117 (2) ◽  
pp. 297-307
Author(s):  
Loverine P Taylor ◽  
Virginia Walbot
Keyword(s):  
Direct Repeat ◽  
Repeat Sequence ◽  
Gene Probe ◽  
Gene Transcript ◽  
Chimeric Transcript ◽  
Purple Pigment ◽  
Isolation And Characterization ◽  
Internal Sequence ◽  
Normal Gene

ABSTRACT We have cloned and sequenced a 1.7-kb Mu element from a Mutator line of maize and compared its structure to Mu1, a 1.4-kb element. With the exception of a 385-bp block of DNA present in the 1.7-kb element, these transposable elements are structurally similar, sharing terminally inverted and internal direct repeated sequences. Derivation of 1.4-kb elements from the 1.7-kb class via deletion of internal sequence is suggested by the finding that a portion of the extra DNA in Mu1.7 is part of a truncated direct repeat sequence in the 1.4-kb element. An abundant poly(A)+ RNA homologous to a portion of this extra DNA is present in several tissues of both Mutator and non-Mutator lines. Analysis of transcripts from an unstable mutant bronze1 (bz) allele containing a Mu1.7 element inserted in an exon of the gene detects three species of poly(A)+ RNA that hybridize to a Bz1 (Bronze) gene probe: the largest contains the entire Mu1.7 element in the Bz1 gene transcript; another appears to be a spliced, chimeric transcript; the smallest is normal size Bz1 mRNA. The latter is most likely encoded by the normal-size alleles detected by Southern analysis of tissue expressing purple pigment, suggesting that normal gene function is restored by excision of the Mu1.7 element.

scholarly journals Synthetic protein-binding DNA sponge as a tool to tune gene expression and mitigate protein toxicity

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xinyi Wan ◽  
Filipe Pinto ◽  
Luyang Yu ◽  
Baojun Wang
Keyword(s):  
Gene Expression ◽  
Gene Regulation ◽  
Protein Binding ◽  
Gene Networks ◽  
Dynamic Range ◽  
Gene Expression Regulation ◽  
Single Layer ◽  
Regulation Mechanism ◽  
Gene Circuits ◽  
Synthetic Dna

AbstractVersatile tools for gene expression regulation are vital for engineering gene networks of increasing scales and complexity with bespoke responses. Here, we investigate and repurpose a ubiquitous, indirect gene regulation mechanism from nature, which uses decoy protein-binding DNA sites, named DNA sponge, to modulate target gene expression in Escherichia coli. We show that synthetic DNA sponges can be designed to reshape the response profiles of gene circuits, lending multifaceted tuning capacities including reducing basal leakage by >20-fold, increasing system output amplitude by >130-fold and dynamic range by >70-fold, and mitigating host growth inhibition by >20%. Further, multi-layer DNA sponges for decoying multiple regulatory proteins provide an additive tuning effect on the responses of layered circuits compared to single-layer sponges. Our work shows synthetic DNA sponges offer a simple yet generalizable route to systematically engineer the performance of synthetic gene circuits, expanding the current toolkit for gene regulation with broad potential applications.

scholarly journals Control of Enzyme IIscr and Sucrose-6-Phosphate Hydrolase Activities in Streptococcus mutans by Transcriptional Repressor ScrR Binding to the cis-Active Determinants of the scr Regulon

2003 ◽  
Vol 185 (19) ◽  
pp. 5791-5799 ◽  
Author(s):  
Bing Wang ◽  
Howard K. Kuramitsu
Keyword(s):  
Streptococcus Mutans ◽  
Transcriptional Repressor ◽  
Direct Repeat ◽  
Dnase I ◽  
Repeat Sequence ◽  
Promoter Regions ◽  
Mobility Shift ◽  
Promoter Sequences ◽  
In The Wild ◽  
Dna Binding Properties

ABSTRACT In Streptococcus mutans, enzyme IIscr and sucrose-6-phosphate hydrolase are two important enzymes in the transport and metabolism of dietary sucrose. The scr regulon of S. mutans is composed of three genes, scrA and scrB, which code for enzyme IIscr and sucrose-6-phosphate hydrolase, respectively, and scrR, which codes for a GalR-LacI-type transcription regulator. It was previously shown that expression of both scrA and scrB is similarly induced by sucrose. Mutation in the scrR gene resulted in increased expression of scrB relative to that in the wild-type strain. In this study, we employed DNA mobility shift and DNase I protection assays with a purified ScrR-histidine tag fusion protein to examine the DNA binding properties of ScrR to the promoter regions of the scrA and scrB genes. The results showed that ScrR bound specifically to the promoter regions of both scrA and scrB. Two regions with high affinity for ScrR in the promoter sequences of the scrA and scrB genes were identified by DNase I protection assays. One, O C, which includes a 20-bp imperfect inverted-repeat sequence, is located between the two promoters, and the other, O B, is located within the scrB promoter region containing a 37-bp imperfect direct-repeat sequence. Mutations of O B and O C resulted in constitutive transcription and expression of both the scrA and scrB genes. Our results indicated that S. mutans coordinates the activities of enzyme IIscr and sucrose-6-phosphate hydrolase by transcriptional repressor ScrR binding to the promoter regions of the scr regulon.

scholarly journals Stability of a Pseudomonas putida KT2440 Bacteriophage-Carried Genomic Island and Its Impact on Rhizosphere Fitness

2012 ◽  
Vol 78 (19) ◽  
pp. 6963-6974 ◽  
Author(s):  
Jose M. Quesada ◽  
María Isabel Soriano ◽  
Manuel Espinosa-Urgel
Keyword(s):  
Pseudomonas Putida ◽  
Genomic Island ◽  
Mobile Element ◽  
Direct Repeat ◽  
Promoter Sequence ◽  
Repeat Sequence ◽  
Genomic Islands ◽  
Pseudomonas Putida Kt2440 ◽  
Bacterial Populations ◽  
Content Type

ABSTRACTThe stability of seven genomic islands ofPseudomonas putidaKT2440 with predicted potential for mobilization was studied in bacterial populations associated with the rhizosphere of corn plants by multiplex PCR. DNA rearrangements were detected for only one of them (GI28), which was lost at high frequency. This genomic island of 39.4 kb, with 53 open reading frames, shows the characteristic organization of genes belonging to tailed phages. We present evidence indicating that it corresponds to the lysogenic state of a functional bacteriophage that we have designated Pspu28. Integrated and rarely excised forms of Pspu28 coexist in KT2440 populations. Pspu28 is self-transmissible, and an excisionase is essential for its removal from the bacterial chromosome. The excised Pspu28 forms a circular element that can integrate into the chromosome at a specific location,attsites containing a 17-bp direct repeat sequence. Excision/insertion of Pspu28 alters the promoter sequence and changes the expression level of PP_1531, which encodes a predicted arsenate reductase. Finally, we show that the presence of Pspu28 in the lysogenic state has a negative effect on bacterial fitness in the rhizosphere under conditions of intraspecific competition, thus explaining why clones having lost this mobile element are recovered from that environment.

EFFICIENT DESIGN OF BINARY TO RNS CONVERTERS

1999 ◽  
Vol 09 (03n04) ◽  
pp. 145-154 ◽  
Author(s):  
P. V. ANANDA MOHAN
Keyword(s):  
Dynamic Range ◽  
Detailed Comparison ◽  
Efficient Design ◽  
Large Dynamic Range ◽  
Recent Interest ◽  
Conversion Time ◽  
Two Stages ◽  
Composite Moduli

In this paper, the choice of composite moduli for realising a large dynamic range RNS is considered. The objective is to reduce the hardware as well as conversion time for Binary to RNS conversion. The proposed designs use two stages of conversion, the first using Periodicity properties of 2x mod mi and second using Binary to RNS conversion architectures proposed in literature due to Alia–Martinelli,1 Capocelli–Giancarlo2 and Stouraits et al.7 Detailed comparison regarding the area requirements and conversion time are presented. The proposed architectures do not need any ROMs for binary to RNS conversion. The results have been extended to the case of even moduli as well due to the recent interest in using one even modulus in certain RNSs.

Conservation of a satellite DNA sequence (SATB) in the tilapiine and haplochromine genome (Pisces: Cichlidae)

Genome ◽  
1993 ◽  
Vol 36 (1) ◽  
pp. 187-194 ◽  
Author(s):  
Jens P. C. Franck ◽  
Jonathan M. Wright
Keyword(s):  
Satellite Dna ◽  
Direct Repeat ◽  
Repeat Sequence ◽  
South American ◽  
Direct Repeats ◽  
Core Motif ◽  
Hindiii Fragment ◽  
Sequence Identity ◽  
Repeat Sequences ◽  
Internal Component

We have cloned and sequenced a 1900-bp EcoRI fragment (SATB) from the tilapiine fish Oreochromis niloticus. The SATB sequence is highly reiterated in the tilapiine genome and organized in long tandem arrays. A 760-bp HindIII fragment, an internal component of SATB, has also been cloned and sequenced from the related tilapiine species Oreochromis hornorum. Hybridization of the radiolabeled 760-bp HindIII repeat detected the presence of the SATB repeat in the genomes of several tilapiine species as well as the haplochromine species Haplochromis (Protomelas) similis. The 760-bp HindIII fragment did not hybridize to genomic DNA of Etroplus maculatus (an Asian cichlid) or to that of Cichlasoma meeki (a South American cichlid). The SATB repeat sequence is 56% AT and constitutes 0.2–5% of the tilapiine genome depending on the species examined. Four imperfect 21-bp direct repeat sequences are present within the cloned 1900-bp EcoRI repeat. Alignment of the four direct repeats from the O. niloticus cloned 1900-bp DNA and the two homologous direct repeats from the O. hornorum 760-bp HindIII repeat revealed a core motif of 11 bp that exhibits 100% sequence identity between all of the direct repeats. The conservation of this motif in the SATB repeat suggests that this sequence may be under selective constraint.Key words: satellite DNA, Cichlidae, direct repeats.

scholarly journals HSP90 Protein Stabilizes Unloaded Argonaute Complexes and Microscopic P-bodies in Human Cells

2010 ◽  
Vol 21 (9) ◽  
pp. 1462-1469 ◽  
Author(s):  
Michael Johnston ◽  
Marie-Claude Geoffroy ◽  
Andrew Sobala ◽  
Ron Hay ◽  
Gyorgy Hutvagner
Keyword(s):  
Gene Regulation ◽  
Small Rna ◽  
Hsp90 Inhibitor ◽  
Gene Repression ◽  
Processing Bodies ◽  
P Bodies ◽  
Protein Levels ◽  
Mirna Level ◽  
Efficient Loading ◽  
Hsp90 Protein

Key components of the miRNA-mediated gene regulation pathway are localized in cytoplasmic processing bodies (P-bodies). Mounting evidence suggests that the presence of microscopic P-bodies are not always required for miRNA-mediated gene regulation. Here we have shown that geldanamycin, a well-characterized HSP90 inhibitor, abolishes P-bodies and significantly reduces Argonaute and GW182 protein levels but does not affect the miRNA level and the efficiency of miRNA-mediated gene repression; however, it significantly impairs siRNA loading and the efficacy of exogenous siRNA. Our data suggests that HSP90 protein chaperones Argonautes before binding RNA and may facilitate efficient loading of small RNA.

scholarly journals Genomic organization of the glycoprotein D gene: Duffy blood group Fya/Fyb alloantigen system is associated with a polymorphism at the 44- amino acid residue

Blood ◽  
1995 ◽  
Vol 85 (3) ◽  
pp. 622-626 ◽  
Author(s):  
S Iwamoto ◽  
T Omi ◽  
E Kajii ◽  
S Ikemoto
Keyword(s):  
Amino Acid ◽  
Blood Group ◽  
Direct Repeat ◽  
Base Change ◽  
Repeat Sequence ◽  
Blood Group System ◽  
Glycoprotein D ◽  
Flanking Sequence ◽  
Duffy Blood Group ◽  
Flanking Region

The Duffy blood group antigen has been characterized by its roles on red blood cells: as a receptor for the malarial parasites and as a promiscuous receptor for chemokine superfamily. Recently, the Duffy blood group associated glycoprotein D (gpFy) cDNA has been cloned (Chaudhuri et al: Proc Natl Acad Sci USA 90:10793, 1993). In this report we describe the organization of genomic DNA coding for the gpFy and elucidate the molecular nature of Fya/b polymorphisms. By a Southern blotting analysis probed with gpFy cDNA, gpFy gene was shown to be composed of three DNA fragments; 1.1-kb Sac I, 1.9-kb EcoRI, and their intervening 47-bp fragments. We cloned the 1.1-kb Sac I and 1.9- kb EcoRI fragments by inverted polymerase chain reaction (IPCR) procedure. The promoter region of the gpFy gene was cloned by IPCR of 1.1-kb Sac I fragment and the 3′ flanking sequence was cloned by IPCR of 1.9 kb EcoRI fragment. The both IPCR products contained on both side the known gpFy cDNA sequence without introns, as expected. Although no TATA or CCAAT boxes are present in the promoter sequence, several transcription factor binding site motifs are contained, including AP-1, HNF-5, TCF-1, ApoE B2, W-element, H-APF-1, and Sp-1. The 3′ flanking region has two additional polyadenylation signals, other than that used in the cDNA, and also has an indirect and a direct repeat sequence clustered with the 5′ flanking region. These facts indicate a possibility that the gpFy gene has been evolved by multiple retrotransposition events. By comparing the coding area of the gpFy gene in 28 Duffy-positive individuals, we elucidated that one base change that results in an amino acid substitution [GA-T(Asp44)-- >GGT(Gly)] is in accordance with the Fya/Fyb polymorphism. This fact proves that the gpFy cDNA and its gene described in this report encode the Duffy blood group system.

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