96 Validation of the candidate mutation responsible for embryonic lethality in Holstein haplotype 2 carriers

2021 ◽  
Vol 33 (2) ◽  
pp. 155
Author(s):  
K. Clark ◽  
J. Cole ◽  
D. Bickhart ◽  
J. Hutchison ◽  
D. Null ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Frameshift Mutation ◽  
Embryonic Lethality ◽  
High Rate ◽  
Reference Sequence ◽  
Future Research ◽  
Wild Type ◽  
Protein Amino Acid ◽  
Cell Stage ◽  
Target Sequences

Holstein haplotype 2 (HH2) is embryonic lethal and carried by 1.21% of the US Holstein population. Using next-generation sequencing, we identified a high-impact frameshift mutation in intraflagellar protein 80 (IFT80) as the putative causal mutation. In bovine embryos, IFT80 expression begins at the 8-cell stage and decreases by the blastocyst stage. We hypothesised that the loss of function of IFT80 early in development causes the lethal phenotype. The aim of this study was to mimic the mutation observed invivo using a CRISPR-Cas9 approach to determine its effect on embryo development. Two guide RNAs (gRNAs) were designed to disrupt exon 11 (Ex11), one before and one after the known IFT80 mutation site, creating a 317-nucleotide (nt) cut to facilitate genotyping. Then, gRNAs annealed to a tracr-Cas9mRNA complex were delivered to 1-cell embryos by microinjection. Each replicate contained control embryos injected with only Cas9mRNA and treated embryos injected with gRNAs targeting IFT80. Embryos from each group were collected at the 8-cell stage for genotyping and gene expression analysis (n=47), or on Day 8 to validate genotypes of embryos left to develop (n=50). DNA sequences containing gRNA target sequences were amplified and visualised on an agarose gel. IFT80 expression was determined in biallelic embryos (n=13) using quantitative PCR and normalized to GAPDH. Primers were designed for the transcript regions before and after gRNAs target sequences, exons 9 and 12, respectively. Expression data were analysed using SAS software (v. 9.4; SAS Institute Inc.) using PROC GLM and LSMEANS to determine expression differences. Biallelic samples (n=9) were Sanger-sequenced (SS) and aligned with the reference sequence to determine exact cut sites. Protein amino acid (AA) sequences were predicted using SS data. Protein models were constructed using the I-Tasser platform, and then aligned and visualised using PyMol 2.4. Biallelic edits showed a significant decrease in exon 12 expression (P<0.05), and no difference in exon 9 compared with controls (P>0.05), indicating that the transcript was severely affected downstream of the edited sites. The reference protein model contained 777 AA, whereas the biallelic sample with the most accurate cut sites yielded a 385-AA protein, indicating that the mutation severely altered protein conformation and possible function. Embryos injected with CRISPR-Cas9 targeting Ex11 arrested at the 8-cell stage and failed to form blastocysts. Day 8 embryos were genotyped (n=24) and 58% were biallelic, 21% were monoallelic, and 21% appeared wild-type. Given the high rate of edits, the observed embryonic arrest is likely due to disruption of IFT80, and wild-type embryos may contain small edits not visible by gel. In conclusion, generation of CRISPR-Cas9 IFT80 knockouts demonstrated that the frameshift mutation in Ex11 results in a seemingly nonfunctional protein that is responsible for the embryonic lethality seen in HH2 carriers. Future research is needed to determine how IFT80 regulates embryonic development. This research was supported by USDA-NIFA National Needs Fellowship, USDA-NIFA AFRI Grant No. 2019-67015-28998.

scholarly journals Quantifying uncertainty of taxonomic placement in DNA barcoding and metabarcoding

2016 ◽  
Author(s):  
Panu Somervuo ◽  
Douglas Yu ◽  
Charles Xu ◽  
Yinqiu Ji ◽  
Jenni Hultman ◽  
...  
Keyword(s):  
Dna Barcoding ◽  
Dna Sequences ◽  
Sequence Data ◽  
Environmental Data ◽  
Reference Sequence ◽  
Future Research ◽  
List Type ◽  
Reference Databases ◽  
Sequence Databases ◽  
Taxonomic Groups

AbstractA crucial step in the use of DNA markers for biodiversity surveys is the assignment of Linnaean taxonomies (species, genus, etc.) to sequence reads. This allows the use of all the information known based on the taxonomic names. Taxonomic placement of DNA barcoding sequences is inherently probabilistic because DNA sequences contain errors, because there is natural variation among sequences within a species, and because reference databases are incomplete and can have false annotations. However, most existing bioinformatics methods for taxonomic placement either exclude uncertainty, or quantify it using metrics other than probability.In this paper we evaluate the performance of a recently proposed probabilistic taxonomic placement method PROTAX by applying it to both annotated reference sequence data as well as unknown environmental data. Our four case studies include contrasting taxonomic groups (fungi, bacteria, mammals, and insects), variation in the length and quality of the barcoding sequences (from individually Sanger-sequenced sequences to short Illumina reads), variation in the structures and sizes of the taxonomies (from 800 to 130 000 species), and variation in the completeness of the reference databases (representing 15% to 100% of the species).Our results demonstrate that PROTAX yields essentially unbiased assessment of probabilities of taxonomic placement, and thus that its quantification of species identification uncertainty is reliable. As expected, the accuracy of taxonomic placement increases with increasing coverage of taxonomic and reference sequence databases, and with increasing ratio of genetic variation among taxonomic levels over within taxonomic levels.Our results show that reliable species-level identification from environmental samples is still challenging, and thus neglecting identification uncertainty can lead to spurious inference. A key aim for future research is the completion and pruning of taxonomic and reference sequence databases, and making these two types of data compatible.

scholarly journals Increased Episomal Replication Accounts for the High Rate of Adaptive Mutation in recD Mutants of Escherichia coli

Genetics ◽  
1999 ◽  
Vol 152 (1) ◽  
pp. 15-30 ◽  
Author(s):  
Patricia L Foster ◽  
William A Rosche
Keyword(s):  
Escherichia Coli ◽  
Mutation Rate ◽  
Copy Number ◽  
Frameshift Mutation ◽  
High Rate ◽  
Adaptive Mutation ◽  
Lacz Gene ◽  
Exonuclease Activity ◽  
Wild Type ◽  
Mutant Cells

Abstract Adaptive mutation has been studied extensively in FC40, a strain of Escherichia coli that cannot metabolize lactose (Lac-) because of a frameshift mutation affecting the lacZ gene on its episome. recD mutants of FC40, in which the exonuclease activity of RecBCD (ExoV) is abolished but its helicase activity is retained, have an increased rate of adaptive mutation. The results presented here show that, in several respects, adaptive mutation to Lac+ involves different mechanisms in recD mutant cells than in wild-type cells. About half of the apparent increase in the adaptive mutation rate of recD mutant cells is due to a RecA-dependent increase in episomal copy number and to growth of the Lac- cells on the lactose plates. The remaining increase appears to be due to continued replication of the episome, with the extra copies being degraded or passed to recD+ recipients. In addition, the increase in adaptive mutation rate in recD mutant cells is (i) dependent on activities of the single-stranded exonucleases, RecJ and ExoI, which are not required for (in fact, slightly inhibit) adaptive mutation in wild-type cells, and (ii) enhanced by RecG, which opposes adaptive mutation in wild-type cells.

Developing a Genetic System in Deinococcus radiodurans for Analyzing Mutations

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 661-668
Author(s):  
Mandy Kim ◽  
Erika Wolff ◽  
Tiffany Huang ◽  
Lilit Garibyan ◽  
Ashlee M Earl ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Deinococcus Radiodurans ◽  
Genetic System ◽  
Base Change ◽  
Base Substitution ◽  
Wild Type ◽  
Base Pairs ◽  
E Coli ◽  
Radiation Induced ◽  
Induced Mutagenesis

Abstract We have applied a genetic system for analyzing mutations in Escherichia coli to Deinococcus radiodurans, an extremeophile with an astonishingly high resistance to UV- and ionizing-radiation-induced mutagenesis. Taking advantage of the conservation of the β-subunit of RNA polymerase among most prokaryotes, we derived again in D. radiodurans the rpoB/Rif r system that we developed in E. coli to monitor base substitutions, defining 33 base change substitutions at 22 different base pairs. We sequenced >250 mutations leading to Rif r in D. radiodurans derived spontaneously in wild-type and uvrD (mismatch-repair-deficient) backgrounds and after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG) and 5-azacytidine (5AZ). The specificities of NTG and 5AZ in D. radiodurans are the same as those found for E. coli and other organisms. There are prominent base substitution hotspots in rpoB in both D. radiodurans and E. coli. In several cases these are at different points in each organism, even though the DNA sequences surrounding the hotspots and their corresponding sites are very similar in both D. radiodurans and E. coli. In one case the hotspots occur at the same site in both organisms.

scholarly journals The Drosophila engrailed and invected Genes: Partners in Regulation, Expression and Function

Genetics ◽  
1996 ◽  
Vol 142 (3) ◽  
pp. 893-906 ◽  
Author(s):  
Elizabeth Gustavson ◽  
Andrew S Goldsborough ◽  
Zehra Ali ◽  
Thomas B Kornberg
Keyword(s):  
Expression Pattern ◽  
Cultured Cells ◽  
Regulatory Region ◽  
Embryonic Lethality ◽  
Wild Type ◽  
Terminal Portion ◽  
Coding Sequence ◽  
Differential Effects ◽  
And Function ◽  
Redundant Functions

Abstract We isolated and characterized numerous engrailed and invected alleles. Among the deficiencies we isolated, a mutant lacking invected sequences was viable and phenotypically normal, a mutant lacking engrailed was an embryo lethal and had slight segmentation defects, and a mutant lacking both engrailed and invected was most severely affected. In seven engrailed alleles, mutations caused translation to terminate prematurely in the central or C-terminal portion of the coding sequence, resulting in embryonic lethality and segmentation defects. Both engrailed and invected expression declined prematurely in these mutant embryos. In wild-type embryos, engrailed and invected are juxtaposed and are expressed in essentially identical patterns. A breakpoint mutant that separates the mgrailed and invected transcription units parceled different aspects of the expression pattern to engrailed or invected. We also found that both genes cause similar defects when expressed ectopically and that the protein products of both genes act to repress transcription in cultured cells. We propose that the varied phenotypes of the engrailed alleles can be explained by the differential effects these mutants have on the combination of engrailed and invected activities, that engrailed and invected share a regulatory region, and that they encode redundant functions.

scholarly journals Somatic production of reactive oxygen species does not predict its production in sperm cells across Drosophila melanogaster lines

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Biz R. Turnell ◽  
Luisa Kumpitsch ◽  
Anne-Cécile Ribou ◽  
Klaus Reinhardt
Keyword(s):  
Reactive Oxygen Species ◽  
Drosophila Melanogaster ◽  
Reactive Oxygen ◽  
Future Research ◽  
Ros Production ◽  
Oxygen Species ◽  
Loss Of Function ◽  
Wild Type ◽  
Ros Scavenging ◽  
Transport Chain

Abstract Objective Sperm ageing has major evolutionary implications but has received comparatively little attention. Ageing in sperm and other cells is driven largely by oxidative damage from reactive oxygen species (ROS) generated by the mitochondria. Rates of organismal ageing differ across species and are theorized to be linked to somatic ROS levels. However, it is unknown whether sperm ageing rates are correlated with organismal ageing rates. Here, we investigate this question by comparing sperm ROS production in four lines of Drosophila melanogaster that have previously been shown to differ in somatic mitochondrial ROS production, including two commonly used wild-type lines and two lines with genetic modifications standardly used in ageing research. Results Somatic ROS production was previously shown to be lower in wild-type Oregon-R than in wild-type Dahomey flies; decreased by the expression of alternative oxidase (AOX), a protein that shortens the electron transport chain; and increased by a loss-of-function mutation in dj-1β, a gene involved in ROS scavenging. Contrary to predictions, we found no differences among these four lines in the rate of sperm ROS production. We discuss the implications of our results, the limitations of our study, and possible directions for future research.

scholarly journals Toward a global reference database of COI barcodes for marine zooplankton

2021 ◽  
Vol 168 (6) ◽  
Author(s):  
Ann Bucklin ◽  
Katja T. C. A. Peijnenburg ◽  
Ksenia N. Kosobokova ◽  
Todd D. O’Brien ◽  
Leocadio Blanco-Bercial ◽  
...  
Keyword(s):  
Species Diversity ◽  
Dna Sequences ◽  
Reference Sequence ◽  
Global Ocean ◽  
Reference Database ◽  
Data Repositories ◽  
Marine Zooplankton ◽  
The North ◽  
Coi Sequences ◽  
Taxonomic Groups

AbstractCharacterization of species diversity of zooplankton is key to understanding, assessing, and predicting the function and future of pelagic ecosystems throughout the global ocean. The marine zooplankton assemblage, including only metazoans, is highly diverse and taxonomically complex, with an estimated ~28,000 species of 41 major taxonomic groups. This review provides a comprehensive summary of DNA sequences for the barcode region of mitochondrial cytochrome oxidase I (COI) for identified specimens. The foundation of this summary is the MetaZooGene Barcode Atlas and Database (MZGdb), a new open-access data and metadata portal that is linked to NCBI GenBank and BOLD data repositories. The MZGdb provides enhanced quality control and tools for assembling COI reference sequence databases that are specific to selected taxonomic groups and/or ocean regions, with associated metadata (e.g., collection georeferencing, verification of species identification, molecular protocols), and tools for statistical analysis, mapping, and visualization. To date, over 150,000 COI sequences for ~ 5600 described species of marine metazoan plankton (including holo- and meroplankton) are available via the MZGdb portal. This review uses the MZGdb as a resource for summaries of COI barcode data and metadata for important taxonomic groups of marine zooplankton and selected regions, including the North Atlantic, Arctic, North Pacific, and Southern Oceans. The MZGdb is designed to provide a foundation for analysis of species diversity of marine zooplankton based on DNA barcoding and metabarcoding for assessment of marine ecosystems and rapid detection of the impacts of climate change.

A Descriptive Analysis of Music Therapy Employment from 2013 to 2019

2020 ◽  
Author(s):  
Jonathon Oden
Keyword(s):  
Music Therapy ◽  
Descriptive Analysis ◽  
American Music ◽  
High Rate ◽  
Future Research ◽  
Full Time ◽  
Music Therapists ◽  
Funding Sources ◽  
Part Time ◽  
Therapy Services

Abstract The purpose of this study was to descriptively analyze music therapy employment data from 2013 to 2019, including years in the field, gender, age, ethnicity, hours worked, jobs created, number of new board-certified music therapists (MT-BCs), funding sources, and wages. A database was created to analyze descriptive data from the 2013–2019 American Music Therapy Association Workforce Analysis Surveys as well as data from the Certification Board for Music Therapists. Results indicate a large portion of music therapists (MTs) have been in the field for five years or less. Though the majority of MTs work full time, there is a high rate of part-time employment. An estimate of the total number of new full-time jobs represented a ratio of 57% of new MT-BCs during the period. Private pay was the most reported funding source for music therapy services. Music therapy wages tended to be higher for those with higher levels of education. Limitations of the study and suggestions for future research are provided.

scholarly journals Molecular analysis of ethyl methanesulfonate-induced reversion of a chromosomally integrated mutant shuttle vector gene in mammalian cells.

1988 ◽  
Vol 8 (10) ◽  
pp. 4185-4189 ◽  
Author(s):  
J A Greenspan ◽  
F M Xu ◽  
R L Davidson
Keyword(s):  
Cell Line ◽  
Cell Lines ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Molecular Mechanisms ◽  
Shuttle Vector ◽  
Ethyl Methanesulfonate ◽  
Wild Type ◽  
Single Base ◽  
Type Sequence

The molecular mechanisms of ethyl methanesulfonate-induced reversion in mammalian cells were studied by using as a target a gpt gene that was integrated chromosomally as part of a shuttle vector. Murine cells containing mutant gpt genes with single base changes were mutagenized with ethyl methanesulfonate, and revertant colonies were isolated. Ethyl methanesulfonate failed to increase the frequency of revertants for cell lines with mutant gpt genes carrying GC----AT transitions or AT----TA transversions, whereas it increased the frequency 50-fold to greater than 800-fold for cell lines with mutant gpt genes carrying AT----GC transitions and for one cell line with a GC----CG transversion. The gpt genes of 15 independent revertants derived from the ethyl methanesulfonate-revertible cell lines were recovered and sequenced. All revertants derived from cell lines with AT----GC transitions had mutated back to the wild-type gpt sequence via GC----AT transitions at their original sites of mutation. Five of six revertants derived from the cell line carrying a gpt gene with a GC----CG transversion had mutated via GC----AT transition at the site of the original mutation or at the adjacent base in the same triplet; these changes generated non-wild-type DNA sequences that code for non-wild-type amino acids that are apparently compatible with xanthine-guanine phosphoribosyltransferase activity. The sixth revertant had mutated via CG----GC transversion back to the wild-type sequence. The results of this study define certain amino acid substitutions in the xanthine-guanine phosphoribosyltransferase polypeptide that are compatible with enzyme activity. These results also establish mutagen-induced reversion analysis as a sensitive and specific assay for mutagenesis in mammalian cells.

scholarly journals Transgenic rescue from embryonic lethality and renal carcinogenesis in the Nihon rat model by introduction of a wild-type Bhd gene

Oncogene ◽  
2005 ◽  
Vol 25 (20) ◽  
pp. 2885-2889 ◽  
Author(s):  
Y Togashi ◽  
T Kobayashi ◽  
S Momose ◽  
M Ueda ◽  
K Okimoto ◽  
...  
Keyword(s):  
Rat Model ◽  
Embryonic Lethality ◽  
Wild Type ◽  
Renal Carcinogenesis ◽  
Nihon Rat

scholarly journals Structural defects of a Pax8 mutant that give rise to congenital hypothyroidism

1999 ◽  
Vol 341 (1) ◽  
pp. 89-93 ◽  
Author(s):  
Gianluca TELL ◽  
Lucia PELLIZZARI ◽  
Gennaro ESPOSITO ◽  
Carlo PUCILLO ◽  
Paolo Emidio MACCHIA ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Congenital Hypothyroidism ◽  
Dna Sequences ◽  
Dna Interaction ◽  
Structural Defects ◽  
Molecular Defect ◽  
Wild Type ◽  
Coding Region ◽  
Induced Fit ◽  
Helical Content

Pax proteins are transcriptional regulators that play important roles during embryogenesis. These proteins recognize specific DNA sequences via a conserved element: the paired domain (Prd domain). The low level of organized secondary structure, in the free state, is a general feature of Prd domains; however, these proteins undergo a dramatic gain in α-helical content upon interaction with DNA (‘induced fit’). Pax8 is expressed in the developing thyroid, kidney and several areas of the central nervous system. In humans, mutations of the Pax8 gene, which are mapped to the coding region of the Prd domain, give rise to congenital hypothyroidism. Here, we have investigated the molecular defects caused by a mutation in which leucine at position 62 is substituted for an arginine. Leu62 is conserved among Prd domains, and contributes towards the packing together of helices 1 and 3. The binding affinity of the Leu62Arg mutant for a specific DNA sequence (the C sequence of thyroglobulin promoter) is decreased 60-fold with respect to the wild-type Pax8 Prd domain. However, the affinities with which the wild-type and the mutant proteins bind to a non-specific DNA sequence are very similar. CD spectra demonstrate that, in the absence of DNA, both wild-type Pax8 and the Leu62Arg mutant possess a low α-helical content; however, in the Leu62Arg mutant, the gain in α-helical content upon interaction with DNA is greatly reduced with respect to the wild-type protein. Thus the molecular defect of the Leu62Arg mutant causes a reduced capability for induced fit upon DNA interaction.

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