In vitro transcription and translation of a full-length cDNA coding for the human ribosomal protein Lia

1993 ◽  
Vol 4 (1) ◽  
pp. 83-88
Author(s):  
Giulia Russo ◽  
Sandro De Falco ◽  
Antonietta Angiolillo ◽  
Concetta Pietropaolo ◽  
Socio A. Ruffo
Keyword(s):  
Ribosomal Protein ◽  
In Vitro Transcription ◽  
Full Length ◽  
Full Length Cdna ◽  
Human Ribosomal Protein

Establishment of Full-length cDNA Clones and an Efficient Oral Infection Model for Feline Coronavirus in Cats

2021 ◽  
Author(s):  
Gang Wang ◽  
Guangli Hu ◽  
Rui Liang ◽  
Jiale Shi ◽  
Xiuxiu Qiu ◽  
...  
Keyword(s):  
Neutralizing Antibodies ◽  
Full Length ◽  
Infection Model ◽  
Type I ◽  
Feline Infectious Peritonitis ◽  
Spike Gene ◽  
Full Length Cdna ◽  
Adult Cats

Feline infectious peritonitis virus (FIPV) is the etiologic agent of feline infectious peritonitis (FIP) and causes fatal disease in cats of almost all ages. Currently, there are no clinically approved drugs or effective vaccines for FIP. Furthermore, the pathogenesis of FIP is still not fully understood. There is an urgent need for an effective infection model of feline infectious peritonitis induced by FIPV. Here, we constructed a field type I FIPV full-length cDNA clone, pBAC-QS, corresponding to the isolated FIPV QS. By replacing the FIPV QS spike gene with the commercially available type II FIPV 79-1146 (79-1146_CA) spike gene, we established and rescued a recombinant virus, designated rQS-79. Moreover, we constructed 79-1146_CA infectious full-length cDNA pBAC-79-1146_CA, corresponding to recombinant FCoV 79-1146_CA (r79-1146_CA). In animal experiments with one- to two-year-old adult cats orally infected with the recombinant virus, rQS-79 induced typical FIP signs and 100% mortality. In contrast to cats infected with rQS-79, cats infected with 79-1146_CA did not show obvious signs. Furthermore, by rechallenging rQS-79 in surviving cats previously infected with 79-1146_CA, we found that there was no protection against rQS-79 with different titers of neutralizing antibodies. However, high titers of neutralizing antibodies may help prolong the cat survival time. Overall, we report the first reverse genetics of virulent recombinant FCoV (causing 100% mortality in adult cats) and attenuated FCoV (causing no mortality in adult cats), which will be powerful tools to study the pathogenesis, antiviral drugs and vaccines for FCoV. Importance Tissue- or cell culture-adapted feline infectious peritonitis virus (FIPV) usually loses pathogenicity. To develop a highly virulent FIPV, we constructed a field isolate type I FIPV full-length clone with the spike gene replaced by the 79-1146 spike gene, corresponding to a virus named rQS-79, which induces high mortality in adult cats. rQS-79 represents the first described reverse genetics system for highly pathogenic FCoV. By further constructing the cell culture-adapted FCoV 79-1146_CA, we obtained infectious clones of virulent and attenuated FCoV. By in vitro and in vivo experiments, we established a model that can serve to study the pathogenic mechanisms of FIPV. Importantly, the wild-type FIPV replicase skeleton of serotype I will greatly facilitate the screening of antiviral drugs, both in vivo and in vitro.

Complete sequence and in vitro expression of a tissue-specific phosphatidylinositol-linked N-CAM isoform from skeletal muscle

Development ◽  
1988 ◽  
Vol 104 (1) ◽  
pp. 165-173 ◽  
Author(s):  
C.H. Barton ◽  
G. Dickson ◽  
H.J. Gower ◽  
L.H. Rowett ◽  
W. Putt ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Amino Acids ◽  
Cell Surface ◽  
Protein Sequence ◽  
Single Copy ◽  
In Vitro Transcription ◽  
Full Length ◽  
Covalent Attachment ◽  
Size Difference

Neural cell adhesion molecules (N-CAMs) are a family of cell surface sialoglycoproteins encoded by a single copy gene. A full-length cDNA clone that encodes a nontransmembrane phosphatidylinositol (PI) linked N-CAM of Mr 125 × 10(3) has been isolated from a human skeletal muscle cDNA library. The deduced protein sequence encodes a polypeptide of 761 amino acids and is highly homologous to the N-CAM isoform in brain of Mr 120 × 10(3). The size difference between the 125 × 10(3). The size difference between the 125 × 10(3) Mr skeletal muscle form and the 120 × 10(3) Mr N-CAM form from brain is accounted for by the insertion of a block of 37 amino acids called MSD1, in the extracellular domain of the muscle form. Transient expression of the human cDNA in COS cells results in cell surface N-CAM expression via a putative covalent attachment to PI-containing phospholipid. Linked in vitro transcription and translation experiments followed by immunoprecipitation with anti-N-CAM antibodies demonstrate that the full-length clone of 761 amino acid coding potential produces a core polypeptide of Mr 110 × 10(3) which is processed by microsomal membranes to yield a 122 × 10(3) Mr species. Taken together, these results demonstrate that the cloned cDNA sequence encodes a lipid-linked, PI-specific phospholipase C releasable surface isoform of N-CAM with core glycopeptide molecular weight corresponding to the authentic muscle 125 × 10(3) Mr N-CAM isoform. This is the first direct correlation of cDNA and deduced protein sequence with a known PI-linked N-CAM isoform from skeletal muscle.

scholarly journals In vitro transcription of infectious RNAs from full-length cDNAs of tobacco mosaic virus

1986 ◽  
Vol 83 (14) ◽  
pp. 5043-5047 ◽  
Author(s):  
T. Meshi ◽  
M. Ishikawa ◽  
F. Motoyoshi ◽  
K. Semba ◽  
Y. Okada
Keyword(s):  
Tobacco Mosaic Virus ◽  
Mosaic Virus ◽  
In Vitro Transcription ◽  
Full Length

Isolation of a full-length cDNA encoding calreticulin from a PCR library of in vitro zygotes of maize

1996 ◽  
Vol 31 (1) ◽  
pp. 23-34 ◽  
Author(s):  
Thomas Dresselhaus ◽  
Christine Hagel ◽  
Horst L�rz ◽  
Erhard Kranz
Keyword(s):  
Full Length ◽  
Full Length Cdna

scholarly journals A novel full-length gene of human ribosomal protein L14.22 related to human glioma

2006 ◽  
Vol 119 (16) ◽  
pp. 1353-1358
Author(s):  
Zhen-yu QI ◽  
Guo-zhen HUI ◽  
Yao LI ◽  
Zong-xiang ZHOU ◽  
Shao-hua GU ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Full Length ◽  
Human Glioma ◽  
Human Ribosomal Protein ◽  
Full Length Gene

scholarly journals In Vitro Analysis of Erythroid Cells Derived from the Culture of Diamond Blackfan Anemia Patient CD34+ Cells

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 744-744
Author(s):  
Kelly A O'Brien ◽  
Stacie M Anderson ◽  
Jason Farrar ◽  
Adrianna Vlachos ◽  
Eva Atsidaftos ◽  
...  
Keyword(s):  
Ribosomal Protein ◽  
Blot Analysis ◽  
Target Genes ◽  
Erythroid Differentiation ◽  
Full Length ◽  
Erythroid Cells ◽  
Ribosomal Protein Genes ◽  
Diamond Blackfan Anemia ◽  
Gata1 Mutation

Abstract Diamond Blackfan anemia (DBA) is a rare, congenital bone marrow failure syndrome characterized by severe macrocytic anemia, usually without perturbation of other hematopoietic lineages. DBA patients are generally diagnosed during infancy or early childhood, have a high frequency of congenital anomalies, and a predisposition to cancer. Approximately 65% of DBA patients have heterozygous mutations or deletions in ribosomal protein genes. Additionally, mutations in the GATA1 gene, which encodes the GATA1 erythroid transcription factor, have been demonstrated in two DBA patients (Sankaran VG et al. J Clin Invest. 122: 2439-43, 2012). The genetic cause of DBA in the remaining 35% of patients is unknown. Despite our knowledge of the genotypes, the mechanism underlying the erythroid failure in DBA is not completely understood. This is largely due to the inability to study primary erythroid cells from DBA patients. To begin to delineate the mechanisms regulating erythroid differentiation in DBA, we developed an in vitro culture system starting with CD34+ progenitor cells isolated from approximately 20 ml of DBA patient peripheral blood collected prior to transfusion at the DBA Registry of North America. Using this system, we have characterized DBA patients with mutations in large (RPL5) and small (RPS17) subunit ribosomal protein genes, one individual with a mutation in the GATA1 gene, and several patients with unknown mutations. At the end of the culture, we routinely obtained 6x107 CD235+ erythroid cells from an initial population of 5x104 control CD34+ progenitor cells. In contrast to control cells, cells from the DBA patients exhibited a significantly reduced growth rate and generated approximately 100-fold fewer CD235+ erythroid cells, with a two day delay in the acquisition of the CD235 marker. Using flow cytometry, we isolated populations of CD41-, CD44+, CD235+ erythroid cells from both control and patient cell cultures from which we extracted RNA. This allowed the first time comparison of mRNA expression in DBA erythroid cells. Protein coding and long non-coding RNA transcripts were compared using Affymetrix GeneChip Human Gene ST Arrays and RNASeq. Compared to controls, CD235+ cells from the patient with the RPL5 mutation showed decreased levels of the GAS5 (growth arrest) and NOP56 (large ribosomal subunit assembly) mRNAs, as well as small nucleolar RNAs. Ingenuity Pathway Analysis (IPA) identified the tRNA charging and RNA Polymerase II assembly pathways as significantly perturbed in this patient. The DBA-associated splice donor mutation in GATA1 results in the exclusive expression of the short form of the GATA1 protein (GATA1s), which lacks the transactivation domain. Western blot analysis of CD235+ control cells showed expression of both full length GATA1 and GATA1s, with the full length protein predominating. In the patient cells with the GATA1 mutation, only the GATA1s protein was expressed and exceeded the combined level of both GATA1 isoforms in the controls. Northern blot analysis demonstrated that the GATA1 mutation did not affect ribosomal RNA processing. The microarray and RNASeq expression profiles of the patient with the GATA1 mutation differed significantly from controls and from that of the patient with the RPL5 mutation. Many known GATA1 target genes including SLC4A1, AHSP, and TRIM10 were down regulated in the CD235+ erythroid cells of the patient with the GATA1mutation compared to control cells, clearly indicating that these genes depend on full length GATA1 for activation. IPA identified the heme biosynthesis pathway as significantly perturbed in this patient and GATA1 as the top regulator. In summary, we have shown that DBA patient cells show decreased proliferative and erythroid differentiation capabilities in vitro. RNA transcript analysis in DBA patient cells has revealed significant differences between DBA patients with a ribosomal protein gene mutation and a mutation in the GATA1 gene. These data delineate multiple signaling pathways or mechanisms involved in DBA and erythroid differentiation. Finally, we have demonstrated that many GATA1 target genes depend on full length GATA1 for activation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Infectious genomic RNA of Rhopalosiphum padi virus transcribed in vitro from a full-length cDNA clone

Virology ◽  
2008 ◽  
Vol 375 (2) ◽  
pp. 401-411 ◽  
Author(s):  
Sandhya Boyapalle ◽  
Randy J. Beckett ◽  
Narinder Pal ◽  
W. Allen Miller ◽  
Bryony C. Bonning
Keyword(s):  
Cdna Clone ◽  
Rhopalosiphum Padi ◽  
Full Length ◽  
Genomic Rna ◽  
Full Length Cdna
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