Forebrain and midbrain regions are deleted in Otx2−/− mutants due to a defective anterior neuroectoderm specification during gastrulation

Development ◽  
1995 ◽  
Vol 121 (10) ◽  
pp. 3279-3290 ◽  
Author(s):  
D. Acampora ◽  
S. Mazan ◽  
Y. Lallemand ◽  
V. Avantaggiato ◽  
M. Maury ◽  
...  
Keyword(s):  
Transcriptional Repression ◽  
Null Allele ◽  
External Layer ◽  
Coding Region ◽  
Coding Sequence ◽  
E Coli ◽  
Homozygous Mutant

We have replaced part of the mouse homeogene Otx2 coding region with the E. coli lacZ coding sequence, thus creating a null allele of Otx2. By 9.5 dpc, homozygous mutant embryos are characterized by the absence of forebrain and midbrain regions. From the early to midstreak stages, endomesodermal cells expressing lacZ fail to be properly localized anteriorly. In the ectodermal layer, lacZ transcription is progressively extinguished, being barely detectable by the late streak stage. These data suggest that Otx2 expression in endomesoderm and ectoderm is required for anterior neuroectoderm specification. In gastrulating heterozygous embryos, a post-transcriptional repression acts on lacZ transcripts in the ectoderm, but not in the external layer, suggesting that different post-transcriptional mechanisms control Otx2 expression in both layers.

scholarly journals A New Type of Fusion Analysis Applicable to Many Organisms: Protein Fusions to the URA3 Gene of Yeast

Genetics ◽  
1987 ◽  
Vol 117 (1) ◽  
pp. 5-12
Author(s):  
Eric Alani ◽  
Nancy Kleckner
Keyword(s):  
Gene Fusion ◽  
Enzyme Assay ◽  
Decarboxylase Activity ◽  
Lacz Gene ◽  
Coding Region ◽  
Ura3 Gene ◽  
Promoter Sequences ◽  
E Coli ◽  
New Type ◽  
Fusion Analysis

ABSTRACT We have made constructs that join the promoter sequences and a portion of the coding region of the Saccharomyces cerevisiae HIS4 and GAL1 genes and the E. coli lacZ gene to the sixth codon of the S. cerevisiae URA3 gene (encodes orotidine-5′-phosphate (OMP) decarboxylase) to form three in frame protein fusions. In each case the fusion protein has OMP decarboxylase activity as assayed by complementation tests and this activity is properly regulated. A convenient cassette consisting of the URA3 segment plus some immediately proximal amino acids of HIS4C is available for making URA3 fusions to other proteins of interest. URA3 fusions offer several advantages over other systems for gene fusion analysis: the URA3 specified protein is small and cytosolic; genetic selections exist to identify mutants with either increased or decreased URA3 function in both yeast (S. cerevisiae and Schizosaccharomyces pombe) and bacteria (Escherichia coli and Salmonella typhimurium); and a sensitive OMP decarboxylase enzyme assay is available. Also, OMP decarboxylase activity is present in mammals, Drosophila and plants, so URA3 fusions may eventually be applicable in these other organisms as well.

scholarly journals Influence of the Leader protein coding region of foot-and-mouth disease virus on virus replication

2013 ◽  
Vol 94 (7) ◽  
pp. 1486-1495 ◽  
Author(s):  
Graham J. Belsham
Keyword(s):  
Disease Virus ◽  
Foot And Mouth Disease ◽  
Mouth Disease ◽  
Initiation Codon ◽  
Coding Region ◽  
Protein Coding ◽  
Coding Sequence ◽  
Bhk Cells ◽  
Leader Protein ◽  
Mouth Disease Virus

The foot-and-mouth disease virus (FMDV) Leader (L) protein is produced in two forms, Lab and Lb, differing only at their amino-termini, due to the use of separate initiation codons, usually 84 nt apart. It has been shown previously, and confirmed here, that precise deletion of the Lab coding sequence is lethal for the virus, whereas loss of the Lb coding sequence results in a virus that is viable in BHK cells. In addition, it is now shown that deletion of the ‘spacer’ region between these two initiation codons can be tolerated. Growth of the virus precisely lacking just the Lb coding sequence resulted in a previously undetected accumulation of frameshift mutations within the ‘spacer’ region. These mutations block the inappropriate fusion of amino acid sequences to the amino-terminus of the capsid protein precursor. Modification, by site-directed mutagenesis, of the Lab initiation codon, in the context of the virus lacking the Lb coding region, was also tolerated by the virus within BHK cells. However, precise loss of the Lb coding sequence alone blocked FMDV replication in primary bovine thyroid cells. Thus, the requirement for the Leader protein coding sequences is highly dependent on the nature and extent of the residual Leader protein sequences and on the host cell system used. FMDVs precisely lacking Lb and with the Lab initiation codon modified may represent safer seed viruses for vaccine production.

scholarly journals Molecular Characterization of Mycoplasma arthritidis Variable Surface Protein MAA2

1998 ◽  
Vol 66 (6) ◽  
pp. 2576-2586 ◽  
Author(s):  
Leigh Rice Washburn ◽  
Keith E. Weaver ◽  
Elizabeth J. Weaver ◽  
Wendy Donelan ◽  
Suhaila Al-Sheboul
Keyword(s):  
Amino Acid ◽  
Signal Peptide ◽  
Dna Sequences ◽  
Surface Protein ◽  
Carboxypeptidase Y ◽  
Coding Region ◽  
Intact Cells ◽  
Mycoplasma Arthritidis ◽  
E Coli ◽  
Variable Surface

Earlier studies implied a role for Mycoplasma arthritidis surface protein MAA2 in cytadherence and virulence and showed that it exhibited both size and phase variability. Here we report the further analysis of MAA2 and the cloning and sequencing of the maa2 gene from two M. arthritidis strains, 158p10p9 and H606, expressing two size variants of MAA2. Triton X-114 partitioning and metabolic labeling with [3H]palmitic acid suggested lipid modification of MAA2. Surface exposure of the C terminus was indicated by cleavage of monoclonal antibody-specific epitopes from intact cells by carboxypeptidase Y. The maa2genes from both strains were highly conserved, consisting largely of six (for 158p10p9) or five (for H606) nearly identical, 264-bp tandem direct repeats. The deduced amino acid sequence predicted a largely hydrophilic, highly basic protein with a 29-amino-acid lipoprotein signal peptide. The maa2 gene was expressed inEscherichia coli from the lacZ promoter of vector pGEM-T. The recombinant product was approximately 3 kDa larger than the native protein, suggesting that the signal peptide was not processed in E. coli. The maa2 gene and upstream DNA sequences were cloned from M. arthritidisclonal variants differing in MAA2 expression state. Expression state correlated with the length of a poly(T) tract just upstream of a putative −10 box. Full-sized recombinant MAA2 was expressed inE. coli from genes derived from both ON and OFF expression variants, indicating that control of expression did not include alterations within the coding region.

Multiple Developmental Functions of the Zinc Finger Gene Krox-20 Are Revealed by Targeted Insertion of the E. coli lacZ Coding Sequence

1998 ◽  
pp. 459-476 ◽  
Author(s):  
Sylvie Schneider-Maunoury ◽  
Piotr Topilko ◽  
Tania Seitanidou ◽  
Giovanni Levi ◽  
Paula Murphy ◽  
...  
Keyword(s):  
Zinc Finger ◽  
Zinc Finger Gene ◽  
Coding Sequence ◽  
E Coli

scholarly journals Hereditary Variation in Platelet Integrin α2β1 Density Is Associated With Two Silent Polymorphisms in the α2 Gene Coding Sequence

Blood ◽  
1997 ◽  
Vol 89 (6) ◽  
pp. 1939-1943 ◽  
Author(s):  
Thomas J. Kunicki ◽  
Marcie Kritzik ◽  
Douglas S. Annis ◽  
Diane J. Nugent
Keyword(s):  
Blood Platelets ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Type I ◽  
Receptor Density ◽  
Coding Region ◽  
Gene Frequencies ◽  
Coding Sequence ◽  
Hereditary Variation ◽  
Donor Population

Abstract The integrin α2β1 is a receptor for collagen that plays a fundamental role in the adhesion of blood platelets to the extracellular matrix. We previously reported that platelet α2β1 levels among randomly selected individuals can vary up to 10-fold and that this correlates with differences in adhesiveness to type-I or type-III collagens. We have now found two linked, allelic polymorphisms within the coding sequence of the α2 gene that correlate with receptor density, TTT/TTC at codon Phe224 and ACA/ACG at codon Thr246. By Southern blot hybridization of specific antisense DNA probes to segments of genomic DNA that encompass each coding region, we have determined the gene frequencies of each allele in a random donor population (n = 65) to be 0.585 (TTC ... ACG) and 0.415 (TTT ... ACA). There is a statistically significant correlation between the alleles TTT ... ACA (codons 224…246) and high receptor density (n = 30; P < .002), whereas the complimentary alleles TTC ... ACG are associated with low receptor density. Heterozygous individuals express intermediate levels of this receptor, and familial studies confirm that these allelic polymorphisms are inherited characteristics. These findings prove that the level of platelet α2β1 is an inherited trait. The molecular basis for receptor density remains to be determined, but our findings establish that these silent alleles within the coding sequence of the α2 gene are linked to the genetic basis for variation in receptor density.

scholarly journals Biochemical and Genetic Studies of the Initiation of Human Rhinovirus 2 RNA Replication: Identification of a cis-Replicating Element in the Coding Sequence of 2Apro

2001 ◽  
Vol 75 (22) ◽  
pp. 10979-10990 ◽  
Author(s):  
Kinga Gerber ◽  
Eckard Wimmer ◽  
Aniko V. Paul
Keyword(s):  
Rna Replication ◽  
Human Rhinovirus ◽  
Viral Sequence ◽  
Coding Region ◽  
Terminal Protein ◽  
Genetic Studies ◽  
Poliovirus Type ◽  
Coding Sequence

ABSTRACT We have previously shown that the RNA polymerase 3Dpolof human rhinovirus 2 (HRV2) catalyzes the covalent linkage of UMP to the terminal protein (VPg) using poly(A) as a template (K. Gerber, E. Wimmer, and A. V. Paul, J. Virol. 75:10969–10978, 2001). The products of this in vitro reaction are VPgpU, VPgpUpU, and VPg-poly(U), the 5′ end of minus-strand RNA. In the present study we used an assay system developed for poliovirus 3Dpol (A. V. Paul, E. Rieder, D. W. Kim, J. H. van Boom, and E. Wimmer, J. Virol. 74: 10359–10370, 2000) to search for a viral sequence or structure in HRV2 RNA that would provide specificity to this reaction. We now show that a small hairpin in HRV2 RNA [cre(2A)], located in the coding sequence of 2Apro, serves as the primary template for HRV2 3Dpol in the uridylylation of HRV2 VPg, yielding VPgpU and VPgpUpU. The in vitro reaction is strongly stimulated by the addition of purified HRV2 3CDpro. Our analyses suggest that HRV2 3Dpol uses a “slide-back” mechanism during synthesis of the VPg-linked precursors. The corresponding cis- replicating RNA elements in the 2CATPase coding region of poliovirus type 1 Mahoney (I. Goodfellow, Y. Chaudhry, A. Richardson, J. Meredith, J. W. Almond, W. Barclay, and D. J. Evans, J. Virol. 74:4590–4600, 2000) and VP1 of HRV14 (K. L. McKnight and S. M. Lemon, RNA 4:1569–1584, 1998) can be functionally exchanged in the assay with cre(2A) of HRV2. Mutations of either the first or the second A in the conserved A1A2A3CA sequence in the loop of HRV2 cre(2A) abolished both viral growth and the RNA's ability to serve as a template in the in vitro VPg uridylylation reaction.

New computational model for miRNA-mediated repression reveals novel regulatory roles of miRNA bindings inside the coding region

2020 ◽  
Author(s):  
Shaked Bergman ◽  
Alon Diament ◽  
Tamir Tuller
Keyword(s):  
Binding Sites ◽  
Supplementary Information ◽  
Coding Region ◽  
Reading Frame ◽  
Coding Sequence ◽  
Elastic Net Regularization ◽  
Physiological Processes ◽  
Ribosome Density ◽  
Weak Binding ◽  
Non Coding Rnas

Abstract Motivation MicroRNAs (miRNAs) are short (∼24nt), non-coding RNAs, which downregulate gene expression in many species and physiological processes. Many details regarding the mechanism which governs miRNA-mediated repression continue to elude researchers. Results We elucidate the interplay between the coding sequence and the 3′UTR, by using elastic net regularization and incorporating translation-related features to predict miRNA-mediated repression. We find that miRNA binding sites at the end of the coding sequence contribute to repression, and that weak binding sites are linked to effective de-repression, possibly as a result of competing with stronger binding sites. Furthermore, we propose a recycling model for miRNAs dissociated from the open reading frame (ORF) by traversing ribosomes, explaining the observed link between increased ribosome density/traversal speed and increased repression. We uncover a novel layer of interaction between the coding sequence and the 3′UTR (untranslated region) and suggest the ORF has a larger role than previously thought in the mechanism of miRNA-mediated repression. Availability and implementation The code is freely available at https://github.com/aescrdni/miRNA_model. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals Identification of the Ovine Keratin-Associated Protein 21-1 Gene and Its Association with Variation in Wool Traits

Animals ◽  
2019 ◽  
Vol 9 (7) ◽  
pp. 450 ◽  
Author(s):  
Li ◽  
Zhou ◽  
Gong ◽  
Zhao ◽  
Wang ◽  
...  
Keyword(s):  
Coding Region ◽  
Gene Encoding ◽  
Coding Sequence ◽  
Sequence Variations ◽  
Associated Proteins ◽  
Wool Yield ◽  
Fleece Weight

Keratin-associated proteins (KAPs) are key constituents of wool and hair fibers. In this study, an ovine KAP gene encoding a HGT-KAP protein was identified. The gene was different from all of the HGT-KAP genes identified in sheep, but was closely related to the human KAP21-1 gene, suggesting that it represented the unidentified ovine KRTAP21-1. Four variants (named A to D) of ovine KRTAP21-1 were found in 360 Merino × Southdown-cross lambs from four sire lines. Three sequence variations were detected among these variants. Two of the sequence variations were located upstream of the coding region and the remaining one was a synonymous variation in the coding sequence. Six genotypes were found in the Merino-cross lambs, with only two of the genotypes (AA and AC) occurring at a frequency of over 5%. Wool from sheep of genotype AA had a higher yield than that from AC sheep (p = 0.014), but tended to have a lower greasy fleece weight (GFW) than that of genotype AC (P = 0.078). This suggests that variation in KRTAP21-1 affects wool yield and the gene may have potential for use as a genetic maker for improving wool yield.

scholarly journals Analysis of a lin-42/period Null Allele Implicates All Three Isoforms in Regulation of Caenorhabditis elegans Molting and Developmental Timing

2016 ◽  
Vol 6 (12) ◽  
pp. 4077-4086 ◽  
Author(s):  
Theresa L B Edelman ◽  
Katherine A McCulloch ◽  
Angela Barr ◽  
Christian Frøkjær-Jensen ◽  
Erik M Jorgensen ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Null Allele ◽  
Postembryonic Development ◽  
Developmental Timing ◽  
Critical Element ◽  
Coding Region ◽  
Protein Coding ◽  
Temporal Regulation ◽  
Larval Stages ◽  
Hypomorphic Alleles

Abstract The Caenorhabditis elegans heterochronic gene pathway regulates the relative timing of events during postembryonic development. lin-42, the worm homolog of the circadian clock gene, period, is a critical element of this pathway. lin-42 function has been defined by a set of hypomorphic alleles that cause precocious phenotypes, in which later developmental events, such as the terminal differentiation of hypodermal cells, occur too early. A subset of alleles also reveals a significant role for lin-42 in molting; larval stages are lengthened and ecdysis often fails in these mutant animals. lin-42 is a complex locus, encoding overlapping and nonoverlapping isoforms. Although existing alleles that affect subsets of isoforms have illuminated important and distinct roles for this gene in developmental timing, molting, and the decision to enter the alternative dauer state, it is essential to have a null allele to understand all of the roles of lin-42 and its individual isoforms. To remedy this problem and discover the null phenotype, we engineered an allele that deletes the entire lin-42 protein-coding region. lin-42 null mutants are homozygously viable, but have more severe phenotypes than observed in previously characterized hypomorphic alleles. We also provide additional evidence for this conclusion by using the null allele as a base for reintroducing different isoforms, showing that each isoform can provide heterochronic and molting pathway activities. Transcript levels of the nonoverlapping isoforms appear to be under coordinate temporal regulation, despite being driven by independent promoters. The lin-42 null allele will continue to be an important tool for dissecting the functions of lin-42 in molting and developmental timing.

Absence of Somatically Acquired JAK1 Mutations in Adult T-Cell Leukemia/Lymphoma.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1921-1921
Author(s):  
Takuro Kameda ◽  
Kotaro Shide ◽  
Haruko Shimoda ◽  
Tomonori Hidaka ◽  
Keiko Katayose ◽  
...  
Keyword(s):  
T Cell ◽  
Mean Value ◽  
Genotype Frequency ◽  
Cell Leukemia ◽  
Coding Region ◽  
Genome Database ◽  
Coding Sequence ◽  
Adult T Cell Leukemia ◽  
The Mean ◽  
Silent Substitutions

Abstract Abstract 1921 Poster Board I-944 Background: Janus kinase 1 (JAK1) plays a critical role in lymphocyte proliferation and differentiation. Somatic JAK1 mutations are found in 18% of adult precursor T acute lymphoblastic leukemias (T-ALL). Some of the mutations were shown to induce the phosphorylation of JAK1 and STAT5 and lead to cytokine-independent proliferation. These data suggest that dysregulation of JAK1 can be involved in the development or progression of T-ALL (Flex et al. J Exp Med. 2008;205:751-758). Adult T-cell leukemia/lymphoma (ATLL) is a type of T-cell neoplasm, and the activation of JAK/STAT is sometimes observed in the tumor cells. Therefore, we investigated JAK1 mutations in ATLL patients. Patients and methods: Twenty Japanese ATLL patients whose percentage of peripheral abnormal lymphocytes was greater than 30% total cell count were sequentially enrolled into the study from 2000 to 2007. Diagnosis of ATLL was made on the basis of clinical features and laboratory characteristics. All cases tested positive for the serum anti-HTLV-1 antibody. The diagnosis was confirmed by observing monoclonal insertion of the HTLV-1 viral genome into leukemia cells by Southern blot hybridization. Peripheral blood mononuclear cells (PBMCs) were isolated and cryopreserved at -80°C. These PBMCs were thawed and genomic DNA was isolated using standard protocol. The entire coding sequence of the JAK1 gene (exons 2 through 25) was amplified by the polymerase chain reaction (PCR) method. The sequence of PCR primers were kindly provided by Dr. Marco Tartaglia (Istituto Superiore di Sanità, Roma, PhD). The nucleotide sequences were determined by fluorescent dye chemistry sequencing and analyzed by sequencing analysis software. By referencing the assembled sequence in the Ensembl genome database, the presence of homozygous mutations was first checked and then candidates for heterozygous mutations or single nucleotide polypeptides (SNPs) on each allele were screened by comparing the ratio of different bases calculated with the height of the peaks seen from sequencing to the reference genome when the ratio was between 0.15 and 1.0. Result: The percentage of abnormal lymphocytes ranged from 30-90%, and the mean value was 55.4%. The mean value of WBC and lymphocyte number was 40.5×109/L and 33.4×109/L, respectively. The mean value of LDH, Ca2+ or sIL-2R was 609 IU/L, 11.4 mg/dL, or 54748 U/mL, respectively. According to Shimoyama criteria (Shimoyama et al. Br J Haematol. 1991;79:428-437), 19 cases were diagnosed as acute-type ATLL, and one case was diagnosed as chronic-type ATLL. The surface markers of all but one abnormal PBMC were CD3+CD4+CD8-CD25+. In that one exception, loss of CD4 expression was observed. We examined the entire coding sequence of the JAK1 gene in 20 ATLL patients and identified no nonsynonymous or nonsense mutations and five types of silent substitutions in 12 cases. All silent substitutions were synonymous SNPs, as determined from referencing the base sequence in the Ensembl genome database. In the ATLL patients examined, the genotype frequency (%) is c546-AA/AG/GG, 97.5/2.5/0; c1590-CC/CT/TT, 97.5/2.5/0; c2049-CC/CT/TT, 50/50/0; c2097-CC/CG/GG, 95/5/0; c2199-AA/AG/GG, 60/40/0. There is no statistical difference in genotype frequency pattern of these SNPs, between the Japanese ATLL patients examined and the general Asian population on the Ensembl database. Conclusion: Mutations in the coding region of JAK1 do not associate with either activation of the JAK/STAT pathway or leukemogenesis in ATLL. We only examined the coding region of JAK1, and the regulatory region of JAK1 remains to be investigated. Further investigation including downstream signaling molecules and inhibitory molecules in the JAK/STAT signaling pathway is necessary to clarify the mechanism contributing to the leukemogenesis of ATLL. Disclosures: No relevant conflicts of interest to declare.

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