Mutational Analysis of the Open Reading Frames in the Transposable Element IS1

Abstract IS1 is one of the smallest transposable elements found in bacteria (768 bp). It contains eight overlapping open-reading-frames (ORFs) greater than 50 codons, designated insA to insG and insB'. To determine which of the ORFs actually code for proteins involved in transposition, we have introduced amber codons into each ORF by site-directed mutagenesis which make neutral changes in the overlapping ORFs. Each mutant IS1 was then tested for its ability to mediate cointegrate formation in Su+ and Su- backgrounds. The mutant elements were also tested for trans-complementation in an IS1-free Salmonella background. Our results show that the products of the insA and insB genes are the only ones essential for cointegrate formation. We suggest that other ORFs may, however, encode accessory proteins.

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Mutational analysis of the 3' open reading frames and the splice junction at nucleotide 3225 of bovine papillomavirus type 1.

Journal of Virology ◽

10.1128/jvi.61.12.3889-3895.1987 ◽

1987 ◽

Vol 61 (12) ◽

pp. 3889-3895 ◽

Cited By ~ 8

Author(s):

P L Hermonat ◽

P M Howley

Keyword(s):

Mutational Analysis ◽

Splice Junction ◽

Open Reading Frames ◽

Bovine Papillomavirus ◽

Reading Frames

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HeT-A, a transposable element specifically involved in "healing" broken chromosome ends in Drosophila melanogaster.

Molecular and Cellular Biology ◽

10.1128/mcb.12.9.3910 ◽

1992 ◽

Vol 12 (9) ◽

pp. 3910-3918 ◽

Cited By ~ 97

Author(s):

H Biessmann ◽

K Valgeirsdottir ◽

A Lofsky ◽

C Chin ◽

B Ginther ◽

...

Keyword(s):

Drosophila Melanogaster ◽

Transposable Element ◽

Sequence Homology ◽

Base Composition ◽

Short Range ◽

Pericentric Heterochromatin ◽

Open Reading Frames ◽

Protein Coding ◽

Reading Frames ◽

Range Variation

Eight terminally deleted Drosophila melanogaster chromosomes have now been found to be "healed." In each case, the healed chromosome end had acquired sequence from the HeT DNA family, a complex family of repeated sequences found only in telomeric and pericentric heterochromatin. The sequences were apparently added by transposition events involving no sequence homology. We now report that the sequences transposed in healing these chromosomes identify a novel transposable element, HeT-A, which makes up a subset of the HeT DNA family. Addition of HeT-A elements to broken chromosome ends appears to be polar. The proximal junction between each element and the broken chromosome end is an oligo(A) tract beginning 54 nucleotides downstream from a conserved AATAAA sequence on the strand running 5' to 3' from the chromosome end. The distal (telomeric) ends of HeT-A elements are variably truncated; however, we have not yet been able to determine the extreme distal sequence of a complete element. Our analysis covers approximately 2,600 nucleotides of the HeT-A element, beginning with the oligo(A) tract at one end. Sequence homology is strong (greater than 75% between all elements studied). Sequence may be conserved for DNA structure rather than for protein coding; even the most recently transposed HeT-A elements lack significant open reading frames in the region studied. Instead, the elements exhibit conserved short-range sequence repeats and periodic long-range variation in base composition. These conserved features suggest that HeT-A elements, although transposable elements, may have a structural role in telomere organization or maintenance.

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Thrombopoietin Production Is Inhibited by a Translational Mechanism

Blood ◽

10.1182/blood.v92.11.4023 ◽

1998 ◽

Vol 92 (11) ◽

pp. 4023-4030 ◽

Cited By ~ 44

Author(s):

Nico Ghilardi ◽

Adrian Wiestner ◽

Radek C. Skoda

Keyword(s):

Untranslated Region ◽

Mutational Analysis ◽

Serum Levels ◽

Open Reading Frames ◽

Differential Splicing ◽

Mrna Isoforms ◽

Major Site ◽

Exon 2 ◽

Promoter Usage ◽

Reading Frames

Abstract Thrombopoietin (TPO) is a lineage-dominant hematopoietic cytokine that regulates megakaryopoiesis and platelet production. The major site of TPO biosynthesis is the liver. Despite easily detectable levels of liver TPO mRNA, the circulating TPO serum levels are very low. We have observed that translation of TPO mRNA is inhibited by the presence of inhibitory elements in the 5′-untranslated region (5′-UTR). Alternative promoter usage and differential splicing generate at least three TPO mRNA isoforms that differ in the composition of their 5′-UTR. Using mutational analysis we show that physiologically the translation of these TPO mRNA isoforms is strongly inhibited by the presence of AUG codons, which define several short open reading frames (ORFs) in the 5′-UTR and suppress efficient initiation at the physiologic start site. The two regularly spliced isoforms, which account for 98% of TPO mRNA, were almost completely inhibited, whereas a rare splice variant that lacks exon 2 can be more efficiently translated. Thus, inhibition of translation of the TPO mRNA is an efficient mechanism to prevent overproduction of this highly potent cytokine.

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Expression, nucleotide sequence and mutational analysis of two open reading frames in the nif gene region of Anabaena sp. strain PCC 7120

MGG Molecular & General Genetics ◽

10.1007/bf00261725 ◽

1990 ◽

Vol 221 (2) ◽

pp. 227-234 ◽

Cited By ~ 43

Author(s):

Dulal Borthakur ◽

Michele Basche ◽

William J. Buikema ◽

Pritty B. Borthakur ◽

Robert Haselkorn

Keyword(s):

Nucleotide Sequence ◽

Mutational Analysis ◽

Open Reading Frames ◽

Gene Region ◽

Anabaena Sp ◽

Nif Gene ◽

Pcc 7120 ◽

Reading Frames

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Thrombopoietin Production Is Inhibited by a Translational Mechanism

Blood ◽

10.1182/blood.v92.11.4023.423k54_4023_4030 ◽

1998 ◽

Vol 92 (11) ◽

pp. 4023-4030 ◽

Cited By ~ 4

Author(s):

Nico Ghilardi ◽

Adrian Wiestner ◽

Radek C. Skoda

Keyword(s):

Untranslated Region ◽

Mutational Analysis ◽

Serum Levels ◽

Open Reading Frames ◽

Differential Splicing ◽

Mrna Isoforms ◽

Major Site ◽

Exon 2 ◽

Promoter Usage ◽

Reading Frames

Thrombopoietin (TPO) is a lineage-dominant hematopoietic cytokine that regulates megakaryopoiesis and platelet production. The major site of TPO biosynthesis is the liver. Despite easily detectable levels of liver TPO mRNA, the circulating TPO serum levels are very low. We have observed that translation of TPO mRNA is inhibited by the presence of inhibitory elements in the 5′-untranslated region (5′-UTR). Alternative promoter usage and differential splicing generate at least three TPO mRNA isoforms that differ in the composition of their 5′-UTR. Using mutational analysis we show that physiologically the translation of these TPO mRNA isoforms is strongly inhibited by the presence of AUG codons, which define several short open reading frames (ORFs) in the 5′-UTR and suppress efficient initiation at the physiologic start site. The two regularly spliced isoforms, which account for 98% of TPO mRNA, were almost completely inhibited, whereas a rare splice variant that lacks exon 2 can be more efficiently translated. Thus, inhibition of translation of the TPO mRNA is an efficient mechanism to prevent overproduction of this highly potent cytokine.

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Sequence and Mutational Analysis of the 6.7-kb Region Containing nodAFEG Genes of Rhizobium sp. Strain N33: Evidence of DNA Rearrangements

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.1997.10.3.401 ◽

1997 ◽

Vol 10 (3) ◽

pp. 401-406 ◽

Cited By ~ 11

Author(s):

Jean Cloutier ◽

Serge Laberge ◽

Hani Antoun

Keyword(s):

Wild Type Strain ◽

Mutational Analysis ◽

Open Reading Frames ◽

Wild Type ◽

Reading Frame ◽

Onobrychis Viciifolia ◽

Intergenic Regions ◽

Astragalus Cicer ◽

Reading Frames ◽

Rhizobium Sp

A 6.7-kb region upstream of nodBC genes in Rhizobium sp. strain N33 was shown to contain the nodAFEG genes and an open reading frame designated orfZ. The open reading frames for these genes contain 591, 282, 1209, 738, and 1,338 nucleotides respectively. Homologues of these genes were found in other rhizobia with the exception of orfZ, for which there was no counterpart found in the GenBank/EMBL database. Tn5 mutagenesis in nodEG and in the intergenic nodG-B region has shown a Nod+ phenotype on their temperate hosts Onobrychis viciifolia and Astragalus cicer. The nodules formed on O. viciifolia plants by these mutants were altered in shape and size. However, on A. cicer there was only a reduction in the number of nodules formed, compared with the wild-type strain. Sequence analysis of the orfZ-nodA and nodG-B intergenic regions indicates the presence of truncated nodD genes.

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Mutational Analysis of Thrombopietin, c-MPL and Jak2 Genes in Essential Thrombothethymia Patients - Identification of Concurrent Mutations of c-MPL and Jak2.

Blood ◽

10.1182/blood.v110.11.4658.4658 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4658-4658

Author(s):

Jianmin Ding ◽

Hirokazu Komatsu ◽

Shinsuke Iida ◽

Asahi Ito ◽

Hiroki Yano ◽

...

Keyword(s):

Somatic Mutations ◽

Mutational Analysis ◽

Myeloproliferative Disorder ◽

Open Reading Frames ◽

Jak2 Mutation ◽

Single Cell Pcr ◽

Activating Mutation ◽

Single Clone ◽

Chronic Myeloproliferative Disorder ◽

Reading Frames

Abstract The molecular pathogenesis in chronic myeloproliferative disorder including essential thrombocythemia (ET) has been recently clarified. We previously identified a novel activating mutation of c-MPL gene (S505N) as a cause of familial ET. The Jak2 mutation (Jak2V617F) is also reported most frequently as a constitutive activating mutation in ET. Here we screened the mutations in the open reading frames of thrombopoietin (TPO), c-MPL and Jak2 in the 26 Japanese (sporadic) ET patients. None had any somatic mutations in TPO. We found Jak2V617F mutation in 7 patients. Interestingly, one of the patients had the c-MPL somatic mutation (W515L). The c-MPL W515L transfectants presented the autonomous STAT5 and MEK1/2 phosphorylation in the absence of the ligand, which show the c-MPL W515L is a constitutive activating mutation for signalling. The concurrent mutations of Jak2V617F and c-MPL W515L are likely to contribute to the pathogenesis of the thrombocythemia in this patient. Now we are focusing on the issue whether the single clone with both of two mutations occupy or the two clones with either Jak2 or c-MPL mutation coexist in the bone marrow of this ET patient (Single cell PCR is on going with the megakaryocytes of this patient).

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Lactococcus lactis LMG2081 Produces Two Bacteriocins, a Nonlantibiotic and a Novel Lantibiotic

Applied and Environmental Microbiology ◽

10.1128/aem.03988-15 ◽

2016 ◽

Vol 82 (8) ◽

pp. 2555-2562 ◽

Cited By ~ 17

Author(s):

Nemanja Mirkovic ◽

Natalija Polovic ◽

Goran Vukotic ◽

Branko Jovcic ◽

Marija Miljkovic ◽

...

Keyword(s):

Lactococcus Lactis ◽

Mutational Analysis ◽

Open Reading Frames ◽

Spectrometric Analysis ◽

Ionization Time ◽

Content Type ◽

Amino Acid Peptide ◽

Directed Mutation ◽

Bacteriocin Producer ◽

Reading Frames

ABSTRACTBacteriocin producers normally possess dedicated immunity systems to protect themselves from their own bacteriocins.Lactococcus lactisstrains LMG2081 and BGBM50 are known as lactococcin G producers. However, BGBM50 was sensitive to LMG2081, which indicated that LMG2081 might produce additional bacteriocins that are not present in BGBM50. Therefore, whole-genome sequencing of the two strains was performed, and a lantibiotic operon (calledlctLMG) was identified in LMG2081 but not in BGBM50. ThelctLMGoperon contains six open reading frames; the first three genes,lmgA,lmgM, andlmgT, are involved in the biosynthesis and export of bacteriocin, while the other three genes,lmgF,lmgE, andlmgG, are involved in lantibiotic immunity. Mutational analysis confirmed that thelctLMGoperon is responsible for the additional antimicrobial activity. Specifically, site-directed mutation within this operon rendered LMG2081 inactive toward BGBM50. Subsequent purification and electrospray ionization–time of flight mass spectrometric analysis confirmed that the lantibiotic bacteriocin called lacticin LMG is exported as a 25-amino-acid peptide. Lacticin LMG is highly similar to the lacticin 481 group. It is interesting that a bacteriocin producer produces two different classes of bacteriocins, whose operons are located in the chromosome and a plasmid.

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DAP5 enables translation re-initiation on structured messenger RNAs

10.1101/2021.01.21.427569 ◽

2021 ◽

Author(s):

Ramona Weber ◽

Leon Kleemann ◽

Insa Hirschberg ◽

Min-Yi Chung ◽

Eugene Valkov ◽

...

Keyword(s):

Cell Fate ◽

Molecular Mechanisms ◽

Mutational Analysis ◽

Ribosome Profiling ◽

Open Reading Frames ◽

Translation Initiation Factor ◽

Initiation Factor ◽

Messenger Rnas ◽

Upstream Open Reading Frames ◽

Reading Frames

SummaryHalf of mammalian transcripts contain short upstream open reading frames (uORFs) that potentially regulate translation of the downstream coding sequence (CDS). The molecular mechanisms governing these events remain poorly understood. Here we find that the non-canonical initiation factor Death-associated protein 5 (DAP5 or eIF4G2) is selectively required for re-initiation at the main CDS following uORF translation. Using ribosome profiling and luciferase-based reporters coupled with mutational analysis we show that DAP5-mediated re-initiation occurs on messenger RNAs (mRNAs) with long, structure-prone 5′ leader sequences and persistent uORF translation. These mRNAs preferentially code for signalling factors such as kinases and phosphatases. We also report that cap/eIF4F- and eIF4A-dependent recruitment of DAP5 to the mRNA facilitates re-initiation by unrecycled post-termination 40S subunits. Our study reveals important mechanistic insights into how a non-canonical translation initiation factor involved in stem cell fate shapes the synthesis of specific signalling factors.

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