scholarly journals Arginine vasopressin secretion with mutants of wild-type and Brattleboro rats AVP gene.

1997 ◽  
Vol 8 (12) ◽  
pp. 1863-1869
Author(s):  
J K Kim ◽  
S N Summer ◽  
W M Wood ◽  
J L Brown ◽  
R W Schrier
Keyword(s):  
Arginine Vasopressin ◽  
Stop Codon ◽  
Bb Rat ◽  
Wild Type ◽  
Coding Region ◽  
Cos Cells ◽  
Coding Regions ◽  
C Terminus ◽  
Type Gene ◽  
Avp Gene

Defects in peptide processing are associated with several disorders, including central diabetes insipidus (CDI). In the Brattleboro (BB) rat with CDI, the mRNA and protein of arginine vasopressin (AVP) are present in the hypothalamus, but no circulating AVP is detectable, thus suggesting a processing defect. The present study examined AVP secretion in cultured COS cells transfected with various constructs from wild-type and mutated Brattleboro AVP gene precursors. The precursor contains three exons encoding for vasopressin (VP), neurophysin (NP), and glycopeptide (GP). The Brattleboro rat has a deletion of a single base, guanine (G), in the NP coding region that leads to a frameshift, resulting in the loss of normal stop codon. The wild-type pcVP (22.0 +/- 5.2 pg/10[-2] U beta-galactosidase [beta-gal]), but not the mutated BB AVP gene pcBB (1.2 +/- 0.4 pg/10[-2] U beta-gal), was associated with AVP secretion from the COS cells as measured by RIA. The wild-type AVP gene without the GP coding region was associated with AVP release greater (47.4 +/- 13.5 pg/10[-2] U beta-gal, n = 5, P < 0.05, versus pcVP) than the pcVP with intact VP, NP, and GP coding regions. However, the wild-type AVP gene with VP coding region alone was not processed and secreted. Normalizing the pcBB total length with the insertion of a stop codon at the site of the normal stop codon was not associated with AVP secretion (3.0 +/- 1.4 pg/10[-2] U beta-gal). However, insertion of a stop codon so that the pcBB length equaled the length of VP and NP coding regions of the wild type was associated with AVP secretion (13.5 +/- 4.0 pg/10[-2] U beta-gal). When a stop codon was inserted into the wild-type NP coding region at the same site as the G deletion in the pcBB, the AVP secretion was significantly lower (15.1 +/- 5.0 pg/10[-2] U beta-gal) than pcVP with VP + NP but no GP coding regions (47.4 +/- 13.5 pg/10[-2] U beta-gal, n = 5, P < 0.05). In summary, (1) both VP and intact NP, but not GP, coding regions are necessary for AVP processing and secretion; (2) decreasing the length of the NP coding region diminishes but does not abolish AVP processing and secretion; and (3) shortening of the pcBB length with a stop codon at a site comparable to wild-type VP + NP allows AVP secretion, albeit less than with wild-type gene precursor. Thus, the CDI in BB rats is caused by the G deletion in NP coding region. This defect leads to abnormalities that contribute to the abnormal AVP processing. Specifically, the frameshift and absence of a stop codon cause a mutated extended C terminus, which, along with the mutated NP, contribute to the abnormal steps of AVP processing, transport, and secretion in the BB rat. These defects no doubt impair the folding and configuration necessary for normal processing of the AVP gene precursor.

Regulatory function of the Saccharomyces cerevisiae RAS C-terminus

1987 ◽  
Vol 7 (7) ◽  
pp. 2309-2315
Author(s):  
M S Marshall ◽  
J B Gibbs ◽  
E M Scolnick ◽  
I S Sigal
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Adenylate Cyclase ◽  
Attachment Site ◽  
Hydrolytic Activity ◽  
Regulatory Function ◽  
Coding Region ◽  
Activating Mutations ◽  
Terminal Domain ◽  
C Terminus ◽  
Type Gene

Activating mutations (valine 19 or leucine 68) were introduced into the Saccharomyces cerevisiae RAS1 and RAS2 genes. In addition, a deletion was introduced into the wild-type gene and into an activated RAS2 gene, removing the segment of the coding region for the unique C-terminal domain that lies between the N-terminal 174 residues and the penultimate 8-residue membrane attachment site. At low levels of expression, a dominant activated phenotype, characterized by low glycogen levels and poor sporulation efficiency, was observed for both full-length RAS1 and RAS2 variants having impaired GTP hydrolytic activity. Lethal CDC25 mutations were bypassed by the expression of mutant RAS1 or RAS2 proteins with activating amino acid substitutions, by expression of RAS2 proteins lacking the C-terminal domain, or by normal and oncogenic mammalian Harvey ras proteins. Biochemical measurements of adenylate cyclase in membrane preparations showed that the expression of RAS2 proteins lacking the C-terminal domain can restore adenylate cyclase activity to cdc25 membranes.

scholarly journals The petD gene is transcribed by functionally redundant promoters in Chlamydomonas reinhardtii chloroplasts.

1994 ◽  
Vol 14 (9) ◽  
pp. 6171-6179 ◽  
Author(s):  
N R Sturm ◽  
R Kuras ◽  
S Büschlen ◽  
W Sakamoto ◽  
K L Kindle ◽  
...  
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Promoter Activity ◽  
Transcriptional Activity ◽  
Transcription Termination ◽  
Cytochrome F ◽  
Direct Repeats ◽  
Wild Type ◽  
Coding Region ◽  
Putative Promoter ◽  
Coding Regions

FUD6, a nonphotosynthetic mutant of Chlamydomonas reinhardtii, was previously found to be deficient in the synthesis of subunit IV of the cytochrome b6/f complex, the chloroplast petD gene product (C. Lemaire, J. Girard-Bascou, F.-A. Wollman, and P. Bennoun, Biochim. Biophys. Acta 851:229-238, 1986). The lesion in FUD6 is a 236-bp deletion between two 11-bp direct repeats in the chloroplast genome. It extends from 82 to 72 bp upstream of the 5' end of wild-type petD mRNA to 156 to 166 bp downstream of the 5' end. Thus, the deletion extends into the putative promoter and 5' untranslated region of petD. No petD mRNA of the normal size can be detected in FUD6 cells, but a low level of a dicistronic message accumulates, which contains the coding regions for subunit IV and cytochrome f, the product of the upstream petA gene. petD transcriptional activity in FUD6 is not significantly altered from the wild-type level. This transcriptional activity was eliminated by petA promoter disruptions, suggesting that it originates at the petA promoter. We conclude that the petD-coding portion of most cotranscripts is rapidly degraded in FUD6, possibly following processing events that generate the 3' end of petA mRNA. A chloroplast transformant was constructed in which only the sequence from -81 to -2 relative to the major 5' end of the petD transcript was deleted. Although this deletion eliminates all detectable petD promoter activity, the transformant grows phototrophically and accumulates high levels of monocistronic petD mRNA. We conclude that the petD gene can be transcribed by functionally redundant promoters. In the absence of a functional petD promoter, a lack of transcription termination allows the downstream petD gene to be cotranscribed with the petA coding region and thereby expressed efficiently.

scholarly journals Molecular Evidence that the Extracellular Cutinase Pbc1 Is Required for Pathogenicity of Pyrenopeziza brassicae on Oilseed Rape

2003 ◽  
Vol 16 (6) ◽  
pp. 545-552 ◽  
Author(s):  
Donghui Li ◽  
Alison M. Ashby ◽  
Keith Johnstone
Keyword(s):  
Oilseed Rape ◽  
Single Copy ◽  
Leaf Spot Disease ◽  
Molecular Evidence ◽  
Wild Type ◽  
In Planta ◽  
Coding Region ◽  
Causal Organism ◽  
Type Gene ◽  
Culture Supernatants

Recent evidence has suggested that cutinase is required for cuticular penetration and, therefore, is essential for pathogenicity of Pyrenopeziza brassicae, the causal organism of light leaf spot disease of oilseed rape and other brassicas. In order to acquire molecular evidence for the role of cutinase in pathogenesis, the single-copy P. brassicae cutinase gene Pbc1 was disrupted by a transformation-mediated approach. Southern hybridization analysis revealed that in one mutant, NH10-1224, the disruption was due to a tandem insertion of two copies of the disruption vector into the 5′ coding region of Pbc1. In contrast to the wild type, no expression of Pbc1 was detected during in planta growth or in cutin-induced mycelium of NH10-1224 and no cutinase activity was detected in culture supernatants from NH10-1224 using pnitrophenyl butyrate as substrate. Scanning electron microscopy of Brassica napus cotyledons infected with wild-type P. brassicae confirmed that entry into the host is by direct penetration of the cuticle. In contrast, the cutinase-deficient mutant NH10-1224 failed to penetrate the cuticular layer and was unable to develop disease symptoms. This evidence is consistent with the hypothesis that Pbc1 is required for P. brassicae to penetrate the plant cuticle. Demonstration that complementation of NH10-1224 with the Pbc1 wild-type gene restores both cutinase activity and pathogenicity will be required to definitively establish that cutinase is required for successful pathogenesis of brassicas by P. brassicae.

scholarly journals Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

2020 ◽  
Author(s):  
Kotchaphorn Mangkalaphiban ◽  
Feng He ◽  
Robin Ganesan ◽  
Chan Wu ◽  
Richard Baker ◽  
...  
Keyword(s):  
Stop Codon ◽  
Regulatory Elements ◽  
Ribosome Profiling ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Coding Region ◽  
Genome Wide ◽  
A Genome ◽  
Stop Codon Readthrough

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3′-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5′ of the stop codon, six nucleotides 3′ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3′-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3′-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 and mRNA secondary structure in the 3′-UTR had milder effects. Additionally, we found low readthrough genes to have shorter 3′-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

scholarly journals Transcriptome-wide investigation of stop codon readthrough in Saccharomyces cerevisiae

PLoS Genetics ◽  
2021 ◽  
Vol 17 (4) ◽  
pp. e1009538
Author(s):  
Kotchaphorn Mangkalaphiban ◽  
Feng He ◽  
Robin Ganesan ◽  
Chan Wu ◽  
Richard Baker ◽  
...  
Keyword(s):  
Stop Codon ◽  
Regulatory Elements ◽  
Ribosome Profiling ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Coding Region ◽  
Genome Wide ◽  
A Genome ◽  
Stop Codon Readthrough

Translation of mRNA into a polypeptide is terminated when the release factor eRF1 recognizes a UAA, UAG, or UGA stop codon in the ribosomal A site and stimulates nascent peptide release. However, stop codon readthrough can occur when a near-cognate tRNA outcompetes eRF1 in decoding the stop codon, resulting in the continuation of the elongation phase of protein synthesis. At the end of a conventional mRNA coding region, readthrough allows translation into the mRNA 3’-UTR. Previous studies with reporter systems have shown that the efficiency of termination or readthrough is modulated by cis-acting elements other than stop codon identity, including two nucleotides 5’ of the stop codon, six nucleotides 3’ of the stop codon in the ribosomal mRNA channel, and stem-loop structures in the mRNA 3’-UTR. It is unknown whether these elements are important at a genome-wide level and whether other mRNA features proximal to the stop codon significantly affect termination and readthrough efficiencies in vivo. Accordingly, we carried out ribosome profiling analyses of yeast cells expressing wild-type or temperature-sensitive eRF1 and developed bioinformatics strategies to calculate readthrough efficiency, and to identify mRNA and peptide features which influence that efficiency. We found that the stop codon (nt +1 to +3), the nucleotide after it (nt +4), the codon in the P site (nt -3 to -1), and 3’-UTR length are the most influential features in the control of readthrough efficiency, while nts +5 to +9 had milder effects. Additionally, we found low readthrough genes to have shorter 3’-UTRs compared to high readthrough genes in cells with thermally inactivated eRF1, while this trend was reversed in wild-type cells. Together, our results demonstrated the general roles of known regulatory elements in genome-wide regulation and identified several new mRNA or peptide features affecting the efficiency of translation termination and readthrough.

Central Diabetes Insipidus Caused by Arginine Vasopressin Gene Mutation: Report of a Novel Mutation and Review of Literature

2020 ◽  
Vol 52 (11) ◽  
pp. 796-802
Author(s):  
Lara L.I. Feldkamp ◽  
Elke Kaminsky ◽  
Tina Kienitz ◽  
Marcus Quinkler
Keyword(s):  
Diabetes Insipidus ◽  
Gene Mutation ◽  
Arginine Vasopressin ◽  
Novel Mutation ◽  
Gene Mutations ◽  
Coding Region ◽  
Exon 2 ◽  
German Family ◽  
Gene Coding ◽  
Avp Gene

AbstractFamilial neurohypophyseal diabetes insipidus (FNDI) is an autosomal dominant hereditary disorder characterized by severe polydipsia and polyuria that usually presents in early childhood. In this study, we describe a new arginine vasopressin (AVP) gene mutation in an ethnic German family with FNDI and provide an overview of disease-associated AVP-gene mutations that are already described in literature. Three members of a German family with neurohypophyseal diabetes insipidus were studied. Isolated DNA from peripheral blood samples was used for mutation analysis by sequencing the whole coding region of AVP-NPII gene. Furthermore, we searched the electronic databases MEDLINE (Pubmed) as well as HGMD, LOVD-ClinVar, db-SNP and genomAD in order to compare our cases to that of other patients with FNDI. Genetic analysis of the patients revealed a novel heterozygote missense mutation in exon 2 of the AVP gene (c.274T>G), which has not yet been described in literature. We identified reports of more than 90 disease-associated mutations in the AVP gene in literature. The novel mutation of the AVP gene seems to cause FNDI in the presented German family. Similar to our newly detected mutation, most mutations causing FNDI are found in exon 2 of the AVP gene coding for neurophysin II. Clinically, it is important to think of FNDI in young children presenting with polydipsia and polyuria.

Molecular Defects of the RHCE Gene in Rh-Deficient Individuals of the Amorph Type

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 639-646 ◽  
Author(s):  
Baya Chérif-Zahar ◽  
Giorgio Matassi ◽  
Virginie Raynal ◽  
Pierre Gane ◽  
Wolfgang Mempel ◽  
...  
Keyword(s):  
Stop Codon ◽  
Premature Stop Codon ◽  
Coding Region ◽  
Protein Protein Interaction ◽  
Molecular Alterations ◽  
Membrane Complex ◽  
C Terminus ◽  
Rh Proteins ◽  
Silent Allele ◽  
Cryptic Splice Sites

Abstract The deficiency of Rh proteins on the red blood cells from individuals of the Rhnull amorph type may be the result of homozygosity for a silent allele at the RH locus. This phenotype is also associated with the lack or reduced expression of glycoproteins (Rh50, CD47, LW, and glycophorin B), which interact with Rh polypeptides to form the multisubunit Rh membrane complex. In this study, we describe two molecular alterations affecting the RHCEgene in two unrelated Rhnull amorph individuals bearing Rh50 and CD47 normal transcripts. The first type of mutation, located at the donor splice-site in intron 4, induced the activation of two cryptic splice-sites within this intron and one such site in exon 4 that all generated aberrant transcripts. The second type of mutation affected the coding region and introduced a frameshift and a premature stop codon resulting in a shorter predicted protein (398 v 417 residues), including a completely different C-terminus of 76 amino acids. This suggests that protein folding and/or protein-protein interaction mediated by the C-terminal domain of the Rh proteins may play a role in the routing and/or stability of the Rh membrane complex.

Molecular Defects of the RHCE Gene in Rh-Deficient Individuals of the Amorph Type

Blood ◽  
1998 ◽  
Vol 92 (2) ◽  
pp. 639-646 ◽  
Author(s):  
Baya Chérif-Zahar ◽  
Giorgio Matassi ◽  
Virginie Raynal ◽  
Pierre Gane ◽  
Wolfgang Mempel ◽  
...  
Keyword(s):  
Stop Codon ◽  
Premature Stop Codon ◽  
Coding Region ◽  
Protein Protein Interaction ◽  
Molecular Alterations ◽  
Membrane Complex ◽  
C Terminus ◽  
Rh Proteins ◽  
Silent Allele ◽  
Cryptic Splice Sites

The deficiency of Rh proteins on the red blood cells from individuals of the Rhnull amorph type may be the result of homozygosity for a silent allele at the RH locus. This phenotype is also associated with the lack or reduced expression of glycoproteins (Rh50, CD47, LW, and glycophorin B), which interact with Rh polypeptides to form the multisubunit Rh membrane complex. In this study, we describe two molecular alterations affecting the RHCEgene in two unrelated Rhnull amorph individuals bearing Rh50 and CD47 normal transcripts. The first type of mutation, located at the donor splice-site in intron 4, induced the activation of two cryptic splice-sites within this intron and one such site in exon 4 that all generated aberrant transcripts. The second type of mutation affected the coding region and introduced a frameshift and a premature stop codon resulting in a shorter predicted protein (398 v 417 residues), including a completely different C-terminus of 76 amino acids. This suggests that protein folding and/or protein-protein interaction mediated by the C-terminal domain of the Rh proteins may play a role in the routing and/or stability of the Rh membrane complex.

scholarly journals Regulatory function of the Saccharomyces cerevisiae RAS C-terminus.

1987 ◽  
Vol 7 (7) ◽  
pp. 2309-2315 ◽  
Author(s):  
M S Marshall ◽  
J B Gibbs ◽  
E M Scolnick ◽  
I S Sigal
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Adenylate Cyclase ◽  
Attachment Site ◽  
Hydrolytic Activity ◽  
Regulatory Function ◽  
Coding Region ◽  
Activating Mutations ◽  
Terminal Domain ◽  
C Terminus ◽  
Type Gene

Activating mutations (valine 19 or leucine 68) were introduced into the Saccharomyces cerevisiae RAS1 and RAS2 genes. In addition, a deletion was introduced into the wild-type gene and into an activated RAS2 gene, removing the segment of the coding region for the unique C-terminal domain that lies between the N-terminal 174 residues and the penultimate 8-residue membrane attachment site. At low levels of expression, a dominant activated phenotype, characterized by low glycogen levels and poor sporulation efficiency, was observed for both full-length RAS1 and RAS2 variants having impaired GTP hydrolytic activity. Lethal CDC25 mutations were bypassed by the expression of mutant RAS1 or RAS2 proteins with activating amino acid substitutions, by expression of RAS2 proteins lacking the C-terminal domain, or by normal and oncogenic mammalian Harvey ras proteins. Biochemical measurements of adenylate cyclase in membrane preparations showed that the expression of RAS2 proteins lacking the C-terminal domain can restore adenylate cyclase activity to cdc25 membranes.

scholarly journals Bovine submaxillary mucin contains multiple domains and tandemly repeated non-identical sequences

1998 ◽  
Vol 331 (1) ◽  
pp. 193-199 ◽  
Author(s):  
Weiping JIANG ◽  
Joseph T. WOITACH ◽  
Ralph L. KEIL ◽  
Veer P. BHAVANANDAN
Keyword(s):  
Amino Acid ◽  
Stop Codon ◽  
Submaxillary Gland ◽  
Northern Analysis ◽  
Coding Region ◽  
Rich Domain ◽  
C Terminus ◽  
Multiple Messages ◽  
Bovine Submaxillary Mucin ◽  
Domain Iii

A number of cDNA fragments coding for bovine submaxillary mucin (BSM) were cloned, and the nucleotide sequence of the largest clone, BSM421, was determined. Two peptide sequences determined from the purified apoBSM were found near the N-terminus of the mucin-coding region of BSM421. This clone does not contain a start or stop codon, but its 3´ end overlaps with the 5´ end of a previously isolated clone, λBSM10. The composite sequence of 1589 amino acid residues consists of five distinct protein domains, which are numbered from the C-terminus. The cysteine-rich domain I can be further divided into a von Willebrand factor type C repeat and a cystine knot. Domains III and V consist of similar repeated peptide sequences with an average of 47 residues. Domains II and IV do not contain such sequences but are similar to domains III and V in being rich in serine and threonine, many of which are predicted to be potential O-glycosylation sites. Domain III also contains two sequences that match the ATP/GTP-binding site motif A (P-loop). Only β-strands and no α-helices are predicted for the partial deduced amino acid sequence. Northern analysis of submaxillary gland RNA with the BSM421 probe detected multiple messages of BSM with sizes from 1.1 to over 10 kb. The tandemly repeated, non-identical peptide sequences of approx. 47 residues in domains III and V of BSM differ from the tandemly repeated, identical 81-residue sequences of pig submaxillary mucin (PSM), although both BSM and PSM contain similar C-terminal domains. In contrast, two peptide sequences of ovine submaxillary mucin are highly similar (86% and 65% identical respectively) to the corresponding sequences in domain V of BSM.

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