scholarly journals Hmga2 deficiency is associated with allometric growth retardation, infertility, and behavioral abnormalities in mice

2021 ◽  
Author(s):  
Mi Ok Lee ◽  
Jingyi Li ◽  
Brian W Davis ◽  
Srijana Upadhyay ◽  
Hadil M Al Muhisen ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Dna Sequences ◽  
Growth Regulation ◽  
Specific Effect ◽  
Craniofacial Development ◽  
Targeted Mutagenesis ◽  
Sperm Chromatin ◽  
Testis Size ◽  
Coding Sequences ◽  
Genomic Deletions

Abstract The high mobility group AT-hook 2 (HMGA2) protein works as an architectural regulator by binding AT-rich DNA sequences to induce conformational changes affecting transcription. Genomic deletions disrupting HMGA2 coding sequences and flanking non-coding sequences cause dwarfism in mice and rabbits. Here, CRISPR/Cas9 was used in mice to generate the Hmga2 null allele that specifically disrupts only the coding sequence. The loss of one or both alleles of Hmga2 resulted in reduced body size of 20% and 60%, respectively, compared to wild-type littermates as well as an allometric reduction in skull length in Hmga2-/- mice. Both male and female Hmga2-/- mice are infertile, whereas Hmga2+/- mice are fertile. Examination of reproductive tissues of Hmga2-/- males revealed a significantly reduced size of testis, epididymis, and seminal vesicle compared to controls, and 70% of knock-out males showed externalized penis, but no cryptorchidism was observed. Sperm analyses revealed severe oligospermia in mutant males and slightly decreased sperm viability, increased DNA damage but normal sperm chromatin compaction. Testis histology surprisingly revealed a normal seminiferous epithelium, despite the significant reduction in testis size. In addition, Hmga2-/- mice showed a significantly reduced exploratory behavior. In summary, the phenotypic effects in mouse using targeted mutagenesis confirmed that Hmga2 is affecting prenatal and postnatal growth regulation, male reproductive tissue development, and presents the first indication that Hmga2 function is required for normal mouse behavior. No specific effect, despite an allometric reduction, on craniofacial development was noted in contrast to previous reports of an altered craniofacial development in mice and rabbits carrying deletions of both coding and non-coding sequences at the 5’part of Hmga2.

scholarly journals SeqEnhDL: sequence-based classification of cell type-specific enhancers using deep learning models

2020 ◽  
Author(s):  
Yupeng Wang ◽  
Rosario B. Jaime-Lara ◽  
Abhrarup Roy ◽  
Ying Sun ◽  
Xinyue Liu ◽  
...  
Keyword(s):  
Neural Network ◽  
Deep Learning ◽  
Dna Sequences ◽  
Cell Types ◽  
Learning Models ◽  
Cell Type ◽  
Coding Sequences ◽  
Sequence Features ◽  
Cell Type Specific ◽  
Different Cell Types

AbstractWe propose SeqEnhDL, a deep learning framework for classifying cell type-specific enhancers based on sequence features. DNA sequences of “strong enhancer” chromatin states in nine cell types from the ENCODE project were retrieved to build and test enhancer classifiers. For any DNA sequence, sequential k-mer (k=5, 7, 9 and 11) fold changes relative to randomly selected non-coding sequences were used as features for deep learning models. Three deep learning models were implemented, including multi-layer perceptron (MLP), Convolutional Neural Network (CNN) and Recurrent Neural Network (RNN). All models in SeqEnhDL outperform state-of-the-art enhancer classifiers including gkm-SVM and DanQ, with regard to distinguishing cell type-specific enhancers from randomly selected non-coding sequences. Moreover, SeqEnhDL is able to directly discriminate enhancers from different cell types, which has not been achieved by other enhancer classifiers. Our analysis suggests that both enhancers and their tissue-specificity can be accurately identified according to their sequence features. SeqEnhDL is publicly available at https://github.com/wyp1125/SeqEnhDL.

scholarly journals Antigen Presentation of mRNA-Based and Virus-Vectored SARS-CoV-2 Vaccines

Vaccines ◽  
2021 ◽  
Vol 9 (8) ◽  
pp. 848
Author(s):  
Ger T. Rijkers ◽  
Nynke Weterings ◽  
Andres Obregon-Henao ◽  
Michaëla Lepolder ◽  
Taru S. Dutt ◽  
...  
Keyword(s):  
Antigen Presentation ◽  
Conformational Changes ◽  
Dna Sequences ◽  
Neutralizing Antibodies ◽  
Large Scale ◽  
Viral Vector ◽  
Adenovirus Vector ◽  
Platelet Factor ◽  
Immune Mediated ◽  
Protein Encoding

Infection with Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) causes Coronavirus Disease 2019 (COVID-19), which has reached pandemic proportions. A number of effective vaccines have been produced, including mRNA vaccines and viral vector vaccines, which are now being implemented on a large scale in order to control the pandemic. The mRNA vaccines are composed of viral Spike S1 protein encoding mRNA incorporated in a lipid nanoparticle and stabilized by polyethylene glycol (PEG). The mRNA vaccines are novel in many respects, including cellular uptake and the intracellular routing, processing, and secretion of the viral protein. Viral vector vaccines have incorporated DNA sequences, encoding the SARS-CoV-2 Spike protein into (attenuated) adenoviruses. The antigen presentation routes in MHC class I and class II, in relation to the induction of virus-neutralizing antibodies and cytotoxic T-lymphocytes, will be reviewed. In rare cases, mRNA vaccines induce unwanted immune mediated side effects. The mRNA-based vaccines may lead to an anaphylactic reaction. This reaction may be triggered by PEG. The intracellular routing of PEG and potential presentation in the context of CD1 will be discussed. Adenovirus vector-based vaccines have been associated with thrombocytopenic thrombosis events. The anti-platelet factor 4 antibodies found in these patients could be generated due to conformational changes of relevant epitopes presented to the immune system.

scholarly journals You're one in a googol: optimizing genes for protein expression

2009 ◽  
Vol 6 (suppl_4) ◽  
Author(s):  
Mark Welch ◽  
Alan Villalobos ◽  
Claes Gustafsson ◽  
Jeremy Minshull
Keyword(s):  
Protein Expression ◽  
Dna Sequences ◽  
Ad Hoc ◽  
Protein Coding ◽  
Vast Number ◽  
Coding Sequences ◽  
Affect Expression ◽  
Continue Work ◽  
Existing Data ◽  
Rational Sequence

A vast number of different nucleic acid sequences can all be translated by the genetic code into the same amino acid sequence. These sequences are not all equally useful however; the exact sequence chosen can have profound effects on the expression of the encoded protein. Despite the importance of protein-coding sequences, there has been little systematic study to identify parameters that affect expression. This is probably because protein expression has largely been tackled on an ad hoc basis in many independent projects: once a sequence has been obtained that yields adequate expression for that project, there is little incentive to continue work on the problem. Synthetic biology may now provide the impetus to transform protein expression folklore into design principles, so that DNA sequences may easily be designed to express any protein in any system. In this review, we offer a brief survey of the literature, outline the major challenges in interpreting existing data and constructing robust design algorithms, and propose a way to proceed towards the goal of rational sequence engineering.

scholarly journals Regulation of Histone Deacetylase 4 Expression by the SP Family of Transcription Factors

2006 ◽  
Vol 17 (2) ◽  
pp. 585-597 ◽  
Author(s):  
Fang Liu ◽  
Nabendu Pore ◽  
Mijin Kim ◽  
K. Ranh Voong ◽  
Melissa Dowling ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Dna Sequences ◽  
Chromatin Immunoprecipitation ◽  
Histone Deacetylases ◽  
Targeted Mutagenesis ◽  
Cellular Functions ◽  
Specific Expression ◽  
Protein Levels ◽  
Histone Deacetylase 4 ◽  
First Time

Histone deacetylases mediate critical cellular functions but relatively little is known about mechanisms controlling their expression, including expression of HDAC4, a class II HDAC implicated in the modulation of cellular differentiation and viability. Endogenous HDAC4 mRNA, protein levels and promoter activity were all readily repressed by mithramycin, suggesting regulation by GC-rich DNA sequences. We validated consensus binding sites for Sp1/Sp3 transcription factors in the HDAC4 promoter through truncation studies and targeted mutagenesis. Specific and functional binding by Sp1/Sp3 at these sites was confirmed with chromatin immunoprecipitation (ChIP) and electromobility shift assays (EMSA). Cotransfection of either Sp1 or Sp3 with a reporter driven by the HDAC4 promoter led to high activities in SL2 insect cells (which lack endogenous Sp1/Sp3). In human cells, restored expression of Sp1 and Sp3 up-regulated HDAC4 protein levels, whereas levels were decreased by RNA-interference-mediated knockdown of either protein. Finally, variable levels of Sp1 were in concordance with that of HDAC4 in a number of human tissues and cancer cell lines. These studies together characterize for the first time the activity of the HDAC4 promoter, through which Sp1 and Sp3 modulates expression of HDAC4 and which may contribute to tissue or cell-line-specific expression of HDAC4.

scholarly journals Kluyveromyces lactis LAC4 Promoter Variants That Lack Function in Bacteria but Retain Full Function in K. lactis

2005 ◽  
Vol 71 (11) ◽  
pp. 7092-7098 ◽  
Author(s):  
Paul A. Colussi ◽  
Christopher H. Taron
Keyword(s):  
Dna Sequences ◽  
Transcription Initiation ◽  
Fluorescent Protein ◽  
Kluyveromyces Lactis ◽  
Targeted Mutagenesis ◽  
Yeast Cells ◽  
Heterologous Proteins ◽  
Gfp Expression ◽  
E Coli ◽  
Cloned Gene

ABSTRACT The strong LAC4 promoter (PLAC4) from Kluyveromyces lactis has been extensively used to drive expression of heterologous proteins in this industrially important yeast. A drawback of this expression method is the serendipitous ability of PLAC4 to promote gene expression in Escherichia coli. This can interfere with the process of assembling expression constructs in E. coli cells prior to their introduction into yeast cells, especially if the cloned gene encodes a protein that is detrimental to bacteria. In this study, we created a series of PLAC4 variants by targeted mutagenesis of three DNA sequences (PBI, PBII, and PBIII) that resemble the E. coli Pribnow box element of bacterial promoters and that reside immediately upstream of two E. coli transcription initiation sites associated with PLAC4. Mutation of PBI reduced the bacterial expression of a reporter protein (green fluorescent protein [GFP]) by ∼87%, whereas mutation of PBII and PBIII had little effect on GFP expression. Deletion of all three sequences completely eliminated GFP expression. Additionally, each promoter variant expressed human serum albumin in K. lactis cells to levels comparable to wild-type PLAC4. We created a novel integrative expression vector (pKLAC1) containing the PLAC4 variant lacking PBI and used it to successfully clone and express the catalytic subunit of bovine enterokinase, a protease that has historically been problematic in E. coli cells. The pKLAC1 vector should aid in the cloning of other potentially toxic genes in E. coli prior to their expression in K. lactis.

SOME HINTS ON OPEN READING FRAME STATISTICS — HOW ORF LENGTH DEPENDS ON SELECTION

1999 ◽  
Vol 10 (04) ◽  
pp. 635-643 ◽  
Author(s):  
AGNIESZKA GIERLIK ◽  
PAWEŁ MACKIEWICZ ◽  
MARIA KOWALCZUK ◽  
STANISŁAW CEBRAT ◽  
MIROSŁAW R. DUDEK
Keyword(s):  
Genetic Code ◽  
Dna Sequences ◽  
Nucleotide Composition ◽  
Open Reading Frames ◽  
Open Reading Frame ◽  
Antisense Strand ◽  
Coding Sequences ◽  
Reading Frame ◽  
Intergenic Sequences ◽  
Reading Frames

Coding sequences of DNA generate Open Reading Frames (ORFs) inside them with much higher frequency than random DNA sequences do, especially in the antisense strand. This is a specific feature of the genetic code. Since coding sequences are selected for their length, the generated ORFs are indirect results of this selection and their length is also influenced by selection. That is why ORFs found in any genome, even much longer ones than those spontaneously generated in random DNA sequences, should be considered as two different sets of ORFs: The first one coding for proteins, the second one generated by the coding ORFs. Even intergenic sequences possess greater capacity for generating ORFs than random DNA sequences of the same nucleotide composition, which seems to be a premise that intergenic sequences were generated from coding sequences by recombinational mechanisms.

scholarly journals Transcribed germline-limited coding sequences in Oxytricha trifallax

2020 ◽  
Author(s):  
Richard V. Miller ◽  
Rafik Neme ◽  
Derek M. Clay ◽  
Jananan S. Pathmanathan ◽  
Michael W. Lu ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Genome Rearrangement ◽  
Life Cycles ◽  
Open Reading Frames ◽  
Developmentally Regulated ◽  
Noncoding Dna ◽  
Protein Coding ◽  
Coding Sequences ◽  
Dna Elimination ◽  
Nuclear Development

AbstractThe germline-soma divide is a fundamental distinction in developmental biology, and different genes are expressed in germline and somatic cells throughout metazoan life cycles. Ciliates, a group of microbial eukaryotes, exhibit germline-somatic nuclear dimorphism within a single cell with two different genomes. The ciliate Oxytricha trifallax undergoes massive RNA-guided DNA elimination and genome rearrangement to produce a new somatic macronucleus (MAC) from a copy of the germline micronucleus (MIC). This process eliminates noncoding DNA sequences that interrupt genes and also deletes hundreds of germline-limited open reading frames (ORFs) that are transcribed during genome rearrangement. Here, we update the set of transcribed germline-limited ORFs (TGLOs) in O. trifallax. We show that TGLOs tend to be expressed during nuclear development and then are absent from the somatic MAC. We also demonstrate that exposure to synthetic RNA can reprogram TGLO retention in the somatic MAC and that TGLO retention leads to transcription outside the normal developmental program. These data suggest that TGLOs represent a group of developmentally regulated protein coding sequences whose gene expression is terminated by DNA elimination.

scholarly journals MELD-DNA: A new tool for capturing protein-DNA binding

2021 ◽  
Author(s):  
Antonio Bauza ◽  
Alberto Perez
Keyword(s):  
Structural Biology ◽  
Conformational Changes ◽  
Dna Sequences ◽  
Structure Prediction ◽  
Binding Mode ◽  
Binding Modes ◽  
Dna Interactions ◽  
Protein Dna Interactions ◽  
Multiple Binding ◽  
New Software Development

Herein we present MELD-DNA, a novel computational approach to address the problem of protein-DNA structure prediction. This method addresses well-known issues hampering current computational approaches to bridge the gap between structural and sequence knowledge, such as large conformational changes in DNA and highly charged electrostatic interaction during binding. MELD- DNA is able to: i) sample multiple binding modes, ii) identify the preferred binding mode from the ensembles, and iii) provide qualitative binding preferences between DNA sequences. We expect the results presented herein will have impact in the field of biophysics (through new software development), structural biology (by complementing DNA structural databases) and supramolecular chemistry (by bringing new insights into protein-DNA interactions).

Making the cut(s): how Cas12a cleaves target and non-target DNA

2019 ◽  
Vol 47 (5) ◽  
pp. 1499-1510 ◽  
Author(s):  
Daan C. Swarts
Keyword(s):  
Conformational Changes ◽  
Dna Sequences ◽  
Dna Cleavage ◽  
Transcriptional Control ◽  
Molecular Mechanisms ◽  
Nucleic Acid Detection ◽  
Target Dna ◽  
Activated State ◽  
Dna Strand ◽  
Cis And Trans

Abstract CRISPR–Cas12a (previously named Cpf1) is a prokaryotic deoxyribonuclease that can be programmed with an RNA guide to target complementary DNA sequences. Upon binding of the target DNA, Cas12a induces a nick in each of the target DNA strands, yielding a double-stranded DNA break. In addition to inducing cis-cleavage of the targeted DNA, target DNA binding induces trans-cleavage of non-target DNA. As such, Cas12a–RNA guide complexes can provide sequence-specific immunity against invading nucleic acids such as bacteriophages and plasmids. Akin to CRISPR–Cas9, Cas12a has been repurposed as a genetic tool for programmable genome editing and transcriptional control in both prokaryotic and eukaryotic cells. In addition, its trans-cleavage activity has been applied for high-sensitivity nucleic acid detection. Despite the demonstrated value of Cas12a for these applications, the exact molecular mechanisms of both cis- and trans-cleavage of DNA were not completely understood. Recent studies have revealed mechanistic details of Cas12a-mediates DNA cleavage: base pairing of the RNA guide and the target DNA induces major conformational changes in Cas12a. These conformational changes render Cas12a in a catalytically activated state in which it acts as deoxyribonuclease. This deoxyribonuclease activity mediates cis-cleavage of the displaced target DNA strand first, and the RNA guide-bound target DNA strand second. As Cas12a remains in the catalytically activated state after cis-cleavage, it subsequently demonstrates trans-cleavage of non-target DNA. Here, I review the mechanistic details of Cas12a-mediated cis- and trans-cleavage of DNA. In addition, I discuss how bacteriophage-derived anti-CRISPR proteins can inhibit Cas12a activity.

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