scholarly journals The cDNA for the ubiquitin-52-amino-acid fusion protein from rat encodes a previously unidentified 60 S ribosomal subunit protein

1996 ◽  
Vol 315 (1) ◽  
pp. 315-321 ◽  
Author(s):  
Kent L. REDMAN ◽  
Glenn W. BURRIS
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Ribosomal Subunit ◽  
Pcr Amplification ◽  
Coding Region ◽  
Consensus Primer ◽  
Reading Frame ◽  
Coding Sequence ◽  
Cdna Sequences ◽  
Ribosomal Subunit Protein

Rat cDNAs for a 52-amino-acid ribosomal protein (CEP52) that is typically formed as a ubiquitin fusion protein, were cloned following reverse transcription and PCR amplification. CEP52 sequence conservation is demonstrated by the similarity of the human and rat cDNA sequences and the identity of the predicted proteins. Amplification of rat cDNA with a primer specific for the 3´ non-coding region of the CEP52 gene, in combination with a consensus primer for the 5´ end of the ubiquitin coding sequence, provided evidence that the rat CEP52 gene is fused to a ubiquitin reading frame. Direct sequence analysis of this PCR product confirmed the in-frame fusion of a ubiquitin coding sequence to the rat CEP52 gene. Antibodies against a synthetic CEP52 peptide were used to show that expressed CEP52 is associated with the 60 S ribosomal subunit, and that it is not linked to ubiquitin. The quantity of CEP52 found in different tissues is quite variable, but appears to correspond to the amount of ribosomes present. Although the human, Arabidopsis thaliana and Nicotiana tabacum CEP52 genes contain introns within the CEP52 coding region, the rat CEP52 coding sequence appears to lack insertions.

scholarly journals Cloning and characterization of a Schistosoma mansoni 1H and 30S clones as two tegumental vaccine candidate antigens.

2003 ◽  
Vol 50 (1) ◽  
pp. 269-278
Author(s):  
Amr M Shabaan ◽  
Magdy M Mohamed ◽  
Mohga S Abdallah ◽  
Hayat M Ibrahim ◽  
Amr M Karim
Keyword(s):  
Amino Acid ◽  
Fusion Protein ◽  
Schistosoma Mansoni ◽  
Molecular Mass ◽  
Vaccine Development ◽  
Complete Sequence ◽  
Cdna Clones ◽  
Western Blots ◽  
Calculated Molecular Mass ◽  
Reading Frame

Two Schistosoma mansoni cDNA clones 30S and 1H were identified by immunoscreening of sporocyst lambdagt11 library and by random sequencing of clones from lambdaZap libraries, respectively. Clone 30S was one of 30 clones identified by an antibody raised against tegument of 3-h schistosomules. The clone was found to encode an 81 amino-acid protein fragment. It was expressed in Escherichia coli as a fusion protein of calculated molecular mass of about 35 kDa with C-terminus of Schistosoma japonicum glutathione-S-transferase (Sj26; about 26 kDa). The recombinant fusion protein was specifically recognized by serum of rabbits immunized with irradiated cercariae. Clone 1H is one of 76 expressed sequence tags derived from an adult worm library. It encodes the complete sequence of a tegumental membrane protein, Sm13. The 104 amino-acid open reading frame encodes a protein with a calculated molecular mass of about 11.9 kDa. Clone 1H was expressed in E. coli as an insoluble fusion protein with Sj26 of about 40 kDa. In Western blots, the fusion protein was recognized by serum from rabbits vaccinated with irradiated cercariae but not by preimmune rabbit sera. The cloning, characterization and expression of those proteins are therefore potentially usefull for vaccine development.

Cloning, characterisation and expression profile of kisspeptin1 and the kisspeptin1 receptor in the hypothalamic–pituitary–ovarian axis of Chinese alligator Alligator sinensis during the reproductive cycle

2020 ◽  
Vol 32 (8) ◽  
pp. 792 ◽  
Author(s):  
Ruidong Zhang ◽  
Haitao Nie ◽  
Shulong Duan ◽  
Peng Yan ◽  
Ali Izaz ◽  
...  
Keyword(s):  
Amino Acid ◽  
Quantitative Polymerase Chain Reaction ◽  
Reproductive Period ◽  
Full Length ◽  
Reading Frame ◽  
Full Length Cdna ◽  
Cdna Sequences ◽  
Acid Protein ◽  
Amino Acid Precursor ◽  
Alligator Sinensis

Kisspeptin1 (Kiss1), a product of the Kiss1 gene, plays an important role in the regulation of reproduction in vertebrates by activating the Kiss1 receptor (Kiss1R) and its coexpression with gonadotrophin-releasing hormone (GnRH) in GnRH neurons. The purpose of this study was to clone the Kiss1 and Kiss1R genes found in the brain of Alligator sinensis and to explore their relationship with reproduction. The full-length cDNA of Kiss1 is 816bp, the open reading frame (ORF) is 417bp and the gene encodes a 138-amino acid precursor protein. The full-length cDNA of Kiss1R is 2348bp, the ORF is 1086bp and the gene encodes a 361-amino acid protein. Quantitative polymerase chain reaction showed that, except for Kiss1R expression in the hypothalamus, the expression of Kiss1 and Kiss1Rduring the reproductive period of A. sinensis was higher than that in the hypothalamus, pituitary gland and ovary during the hibernation period. The changes in GnRH2 mRNA in the hypothalamus were similar to those of GnRH1 and peaked during the reproductive period. This study confirms the existence of Kiss1 and Kiss1R in A. sinensis and the findings strongly suggest that Kiss1 and Kiss1R may participate in the regulation of GnRH secretion in the hypothalamus of alligators during the reproductive period. Furthermore, this is the first report of the full-length cDNA sequences of Kiss1 and Kiss1R in reptiles.

scholarly journals Cloning of glycoprotein IIIa cDNA from human erythroleukemia cells and localization of the gene to chromosome 17

Blood ◽  
1988 ◽  
Vol 72 (2) ◽  
pp. 593-600 ◽  
Author(s):  
JP Rosa ◽  
PF Bray ◽  
O Gayet ◽  
GI Johnston ◽  
RG Cook ◽  
...  
Keyword(s):  
Endothelial Cell ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Cdna Sequence ◽  
Chromosome 17 ◽  
Coding Region ◽  
Membrane Glycoproteins ◽  
Cdna Sequences ◽  
Hel Cells ◽  
Adhesive Proteins

Abstract Platelet aggregation requires the binding of adhesive proteins such as fibrinogen to the heterodimer of membrane glycoproteins IIb (GPIIb) and IIIa (GPIIIa). Human erythroleukemia (HEL) cells synthesize both GPIIb and GPIIIa. Using poly(A+) RNA purified from HEL cells, we constructed a cDNA library in the lambda gt10 phage vector. This library was screened with a 38mer oligonucleotide derived from a platelet GPIIIa peptide, and three overlapping cDNAs were isolated. The three inserts encompassed 3.5 kilobases (kb), including the entire coding region of mature GPIIIa (2,286 basepairs, bp) and 1.3 kb of 3′ untranslated sequence. All 222 residues determined directly from platelet GPIIIa tryptic peptides exactly matched the HEL cell-deduced amino acid sequence. The HEL cell sequence matched a previously reported endothelial cell cDNA sequence except for eight nucleotides. Five of these nucleotide differences were silent changes consistent with genetic polymorphisms. The other three differences resulted in changes in the deduced amino acid sequence of GPIIIa; reexamination of the endothelial cell cDNA sequence in these three areas revealed that it is actually identical to the HEL cell sequence. The virtual identity of the endothelial and HEL cell cDNA sequences provides direct evidence that GPIIIa is a subunit common to cell-adhesion receptors present in more than one cell type. We localized the gene for GPIIIa to chromosome 17, the same chromosome to which we had previously mapped the gene for GPIIb.

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.

Cloning and expression of caprine KIT gene and associations of polymorphisms with litter size

2016 ◽  
Vol 56 (10) ◽  
pp. 1579 ◽  
Author(s):  
X. P. An ◽  
J. X. Hou ◽  
T. Y. Gao ◽  
B. Y. Cao
Keyword(s):  
Amino Acid ◽  
Litter Size ◽  
Bos Taurus ◽  
Homo Sapiens ◽  
Ovis Aries ◽  
Cloning And Expression ◽  
Nucleotide Polymorphisms ◽  
Coding Region ◽  
Reading Frame ◽  
Kit Gene

The full coding region of KIT mRNA was cloned from the caprine ovary. The results showed the caprine KIT cDNA (GenBank accession number KF364483) contained a 2925-bp open reading frame encoding a protein with 974 amino acid residues. BLAST analysis revealed that the caprine KIT protein had high similarity with that of four species: Ovis aries (99%), Bos taurus (99%), Sus scrofa (94%) and Homo sapiens (90%). The KIT mRNA expression pattern showed that KIT mRNA was expressed highly in kidney, ovary, uterus and breast. Two single nucleotide polymorphisms (g.88430T > A and g.120466G > A) in the caprine KIT gene were detected by PCR–restriction fragment length polymorphism (RFLP) and DNA sequencing in 735 goats of Xinong Saanen, Guanzhong and Boer breeds. The g.88430T > A mutation was a missense mutation (Tyr > Asn at position 409 amino acid of KIT). The association study has been done by jointly analysing all data in one analysis. The result showed that individuals with TT and TA genotypes had their litter size increased by 0.11 and 0.09, respectively, compared with those with AA genotype at the g.88430T > A locus for three goat breeds (P < 0.05). Further analysis revealed that combined genotype TTAA was better than the others for litter size in three goat breeds. Therefore, the biochemical and physiological functions, together with the results obtained in our investigation, suggest that the KIT gene could serve as a genetic marker for litter size in goat breeding.

scholarly journals cDNA nucleotide sequence encoding the ZPC protein of Australian hydromyine rodents: a novel sequence of the putative sperm-combining site within the family Muridae

Zygote ◽  
2002 ◽  
Vol 10 (4) ◽  
pp. 291-299 ◽  
Author(s):  
Christine A. Swann ◽  
Rory M. Hope ◽  
William G. Breed
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Amino Acid Sequence ◽  
Cdna Sequence ◽  
Amino Acid Sequence Identity ◽  
Sequence Data ◽  
Coding Region ◽  
Cdna Sequences ◽  
Sequence Identity ◽  
Murid Rodents

This comparative study of the cDNA sequence of the zona pellucida C (ZPC) glycoprotein in murid rodents focuses on the nucleotide and amino acid sequence of the putative sperm-combining site. We ask the question: Has divergence evolved in the nucleotide sequence of ZPC in the murid rodents of Australia? Using RT-PCR and (RACE) PCR, the complete cDNA coding region of ZPC in the Australian hydromyine rodents Notomys alexis and Pseudomys australis, and a partial cDNA sequence from a third hydromyine rodent, Hydromys chrysogaster, has been determined. Comparison between the cDNA sequences of the hydromyine rodents reveals that the level of amino acid sequence identity between N. alexis and P. australis is 96%, whereas that between the two species of hydromyine rodents and M. musculus and R. norvegicus is 88% and 87% respectively. Despite being reproductively isolated from each other, the three species of hydromyine rodents have a 100% level of amino acid sequence identity at the putative sperm-combining site. This finding does not support the view that this site is under positive selective pressure. The sequence data obtained in this study may have important conservation implications for the dissemination of immunocontraception directed against M. musculus using ZPC antibodies.

scholarly journals Evidence for Production of a New Lantibiotic (Butyrivibriocin OR79A) by the Ruminal Anaerobe Butyrivibrio fibrisolvensOR79: Characterization of the Structural Gene Encoding Butyrivibriocin OR79A

1999 ◽  
Vol 65 (5) ◽  
pp. 2128-2135 ◽  
Author(s):  
M. L. Kalmokoff ◽  
D. Lu ◽  
M. F. Whitford ◽  
R. M. Teather
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Gel Filtration ◽  
Culture Fluid ◽  
Northern Blotting ◽  
Coding Region ◽  
Gene Encoding ◽  
Butyrivibrio Fibrisolvens ◽  
Reading Frame ◽  
Significant Homology

ABSTRACT The ruminal anaerobe Butyrivibrio fibrisolvens OR79 produces a bacteriocin-like activity demonstrating a very broad spectrum of activity. An inhibitor was isolated from spent culture fluid by a combination of ammonium sulfate and acidic precipitations, reverse-phase chromatography, and high-resolution gel filtration. N-terminal analysis of the isolated inhibitor yielded a 15-amino-acid sequence (G-N/Q-G/P-V-I-L-X-I-X-H-E-X-S-M-N). Two different amino acid residues were detected in the second and third positions from the N terminus, indicating the presence of two distinct peptides. A gene with significant homology to one combination of the determined N-terminal sequence was cloned, and expression of the gene was confirmed by Northern blotting. The gene (bvi79A) encoded a prepeptide of 47 amino acids and a mature peptide, butyrivibriocin OR79A, of 25 amino acids. Significant sequence homology was found between this peptide and previously reported lantibiotics containing the double-glycine leader peptidase processing site. Immediately downstream of bvi79Awas a second, partial open reading frame encoding a peptide with significant homology to proteins which are believed to be involved in the synthesis of lanthionine residues. These findings indicate that the isolated inhibitory peptides represent new lantibiotics. Results from both total and N-terminal amino acid sequencing indicated that the second peptide was identical to butyrivibriocin OR79A except for amino acid substitutions in positions 2 and 3 of the mature lantibiotic. Only a single coding region was detected when restriction enzyme digests of total DNA were probed either with an oligonucleotide based on the 5′ region of bvi79A or with degenerate oligonucleotides based on the predicted sequence of the second peptide.

scholarly journals Translation Initiation from Conserved Non-AUG Codons Provides Additional Layers of Regulation and Coding Capacity

mBio ◽  
2017 ◽  
Vol 8 (3) ◽  
Author(s):  
Ivaylo P. Ivanov ◽  
Jiajie Wei ◽  
Stephen Z. Caster ◽  
Kristina M. Smith ◽  
Audrey M. Michel ◽  
...  
Keyword(s):  
Gene Expression ◽  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Neurospora Crassa ◽  
Open Reading Frames ◽  
Reading Frame ◽  
Coding Sequence ◽  
Cell Extracts ◽  
Upstream Open Reading Frames ◽  
Reading Frames

ABSTRACT Neurospora crassa cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying transcription activators that drive the transcriptional response to amino acid limitation. The cpc-1 mRNA contains two upstream open reading frames (uORFs) in its >700-nucleotide (nt) 5′ leader, and its expression is controlled at the level of translation in response to amino acid starvation. We used N. crassa cell extracts and obtained data indicating that cpc-1 uORF1 and uORF2 are functionally analogous to GCN4 uORF1 and uORF4, respectively, in controlling translation. We also found that the 5′ region upstream of the main coding sequence of the cpc-1 mRNA extends for more than 700 nucleotides without any in-frame stop codon. For 100 cpc-1 homologs from Pezizomycotina and from selected Basidiomycota, 5′ conserved extensions of the CPC1 reading frame are also observed. Multiple non-AUG near-cognate codons (NCCs) in the CPC1 reading frame upstream of uORF2, some deeply conserved, could potentially initiate translation. At least four NCCs initiated translation in vitro . In vivo data were consistent with initiation at NCCs to produce N-terminally extended N. crassa CPC1 isoforms. The pivotal role played by CPC1, combined with its translational regulation by uORFs and NCC utilization, underscores the emerging significance of noncanonical initiation events in controlling gene expression. IMPORTANCE There is a deepening and widening appreciation of the diverse roles of translation in controlling gene expression. A central fungal transcription factor, the best-studied example of which is Saccharomyces cerevisiae GCN4, is crucial for the response to amino acid limitation. Two upstream open reading frames (uORFs) in the GCN4 mRNA are critical for controlling GCN4 synthesis. We observed that two uORFs in the corresponding Neurospora crassa cpc-1 mRNA appear functionally analogous to the GCN4 uORFs. We also discovered that, surprisingly, unlike GCN4, the CPC1 coding sequence extends far upstream from the presumed AUG start codon with no other in-frame AUG codons. Similar extensions were seen in homologs from many filamentous fungi. We observed that multiple non-AUG near-cognate codons (NCCs) in this extended reading frame, some conserved, initiated translation to produce longer forms of CPC1, underscoring the significance of noncanonical initiation in controlling gene expression.

scholarly journals Gene Cloning and Tissue Expression Analysis of a PR-5 Thaumatin-Like Protein in Phellinus weirii-Infected Douglas-Fir

2004 ◽  
Vol 94 (11) ◽  
pp. 1235-1243 ◽  
Author(s):  
Arezoo Zamani ◽  
Rona N. Sturrock ◽  
Abul K. M. Ekramoddoullah ◽  
Jun Jun Liu ◽  
Xueshu Yu
Keyword(s):  
Amino Acid ◽  
Immunoblot Analysis ◽  
Tissue Expression ◽  
Douglas Fir ◽  
Coding Region ◽  
General Role ◽  
Peptide Cleavage ◽  
Reading Frame ◽  
Tissue Expression Analysis ◽  
Phellinus Weirii

In western North America, Douglas-fir (Pseudotsuga menziesii) is the most economically important conifer species susceptible to laminated root rot caused by Phellinus weirii. While attempting to internally sequence an endochitinase found to be up-regulated in P. weirii-infected Douglas-fir roots, we obtained overlapping peptide fragments showing 28% similarity with a PR-5 thaumatin-like protein (TLP) designated PmTLP (Pm for Pseudotsuga menziesi). A rabbit polyclonal antibody was reared against a synthetic peptide composed of a 29-amino-acid-long, conserved, internal sequence of PmTLP and purified by immunoaffinity. Western immunoblot analysis of infected roots of 24-year-old coastalfir showed significantly higher amounts of PmTLP (P < 0.01) closest to the point of P. weirii inoculation and infection than in uninfected regions of the same root. The antibody was also used to screen for PmTLP in roots of 25-year-old interior Douglas-firs naturally infected with a related pathogen, Armillaria ostoyae, and results showed significantly higher levels of PmTLP in bark tissues adjacent to infection (P < 0.05) than in uninfected tissue. Using polymerase chain reaction (PCR)-based cloning, the cDNA of PmTLP was shown to have a 702-bp open reading frame with a signal peptide cleavage site at 155 bp corresponding to a 29-amino-acid-long residue prior to the start of the N-terminal. Based on the deduced amino acid sequence, the molecular mass of the putative PmTLP was calculated to be 21.0 kDa with an isoelectric point of 3.71. Alignment analysis of PmTLP cDNA with a representative genomic DNA PCR sequence showed presence of one intron of variable size, within the coding region. The induction of PmTLP at the site of root infection and its presence in needle tissue suggests a general role for this protein in adaptation to stress and may be part of an integrated defense response initiated by the host to impede further pathogen spread.

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