A Transcriptionally Active Human Type II Gonadotropin-Releasing Hormone Receptor Gene Homolog Overlaps Two Genes in the Antisense Orientation on Chromosome 1q.12

GnRH-II peptide hormone exhibits complete sequence conservation across vertebrate species, including man. Type-II GnRH receptor genes have been characterized recently in nonhuman primates, but the human receptor gene homolog contains a frameshift, a premature stop codon (UGA), and a 3′ overlap of the RBM8A gene on chromosome 1q.12. A retrotransposed pseudogene, RBM8B, retains partial receptor sequence. In this study, bioinformatics show that the human receptor gene promoter overlaps the peroxisomal protein11-β gene promoter and the premature UGA is positionally conserved in chimpanzee. A CGA [arginine (Arg)] occurs in porcine DNA, but UGA is shifted one codon to the 5′ direction in bovine DNA, suggesting independent evolution of premature stop codons. In contrast to marmoset tissue RNA, exon- and strand-specific probes are required to distinguish differently spliced human receptor gene transcripts in cell lines (HP75, IMR-32). RBM8B is not transcribed. Sequencing of cDNAs for spliced receptor mRNAs showed no evidence for alteration of the premature UGA by RNA editing, but alternative splicing circumvents the frameshift to encode a two-membrane-domain protein before this UGA. A stem-loop motif resembling a selenocysteine insertion sequence and a potential alternative translation initiation site might enable expression of further proteins involved in interactions within the GnRH system.

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A Single Base Pair Mutation Encoding a Premature Stop Codon in the MIS Type II Receptor Is Responsible for Canine Persistent Mullerian Duct Syndrome

Journal of Andrology ◽

10.2164/jandrol.108.005736 ◽

2008 ◽

Vol 30 (1) ◽

pp. 46-56 ◽

Cited By ~ 32

Author(s):

X. Wu ◽

S. Wan ◽

S. Pujar ◽

M. E. Haskins ◽

D. H. Schlafer ◽

...

Keyword(s):

Base Pair ◽

Stop Codon ◽

Premature Stop Codon ◽

Mullerian Duct ◽

Type Ii ◽

Single Base ◽

Müllerian Duct ◽

Persistent Mullerian Duct Syndrome ◽

Single Base Pair ◽

Duct Syndrome

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91 DISRUPTION OF TET1 DURING PORCINE EMBRYOGENESIS USING CRISPR/Cas9 SYSTEM

Reproduction Fertility and Development ◽

10.1071/rdv29n1ab91 ◽

2017 ◽

Vol 29 (1) ◽

pp. 153

Author(s):

K. Uh ◽

J. Ryu ◽

C. Ray ◽

K. Lee

Keyword(s):

Embryo Development ◽

Translation Initiation ◽

Stop Codon ◽

Premature Stop Codon ◽

Initiation Site ◽

Translation Initiation Site ◽

Epigenetic Marks ◽

Hydroxymethyl Cytosine ◽

Biallelic Mutations

Ten-eleven translocation (TET) enzymes catalyse oxidation of 5-methylcytosine to 5-hydroxymethyl cytosine. This TET-mediated conversion of 5-methylcytosine to 5-hydroxymethyl cytosine is implicated in initiating the DNA demethylation process, observed post-fertilization. Three members (TET1–3) of the TET family are differentially expressed during embryo development and appear to have different roles. Previous studies in mice suggest that TET1 is a key regulator in maintaining pluripotency in embryonic stem cells by managing epigenetic marks such as DNA methylation. This would imply that TET1 should be a regulator of epigenetic marks during embryo development, although this has not been demonstrated. Previously, we have cloned porcine TET1 from blastocysts (GenBank accession number KC137683) and demonstrated that the level of TET1 (mRNA and protein) was high in blastocysts. The protein level was greater in the inner cell mass compared with the trophectoderm. In this study, we generated TET1 knockout porcine embryos using CRISPR/Cas9 system to study the role of TET1 in controlling epigenetic marks during porcine embryo development. First, 2 sgRNA, immediately downstream of the presumable translation initiation site, were designed and synthesised; location of the sgRNA were nucleotide position at 2 to 21 bp and 23 to 42 bp, respectively (KC137683). Then, sgRNA (10 ng μL−1 each) and Cas9 mRNA (20 ng μL−1) were injected into the cytoplasm of IVF zygotes, and Day 7 blastocysts were genotyped. All embryos carried mutations on both alleles of TET1 (10/10), one homozygous and 9 biallelic mutations. However, immunocytochemistry analysis of other CRISPR/Cas9 injected embryos revealed that TET1 was not removed (10/10), indicating that the sgRNA may have not introduced a premature stop codon 3′ to the presumable translation initiation site. Therefore, 2 new sgRNA were designed to generate a premature stop codon at the 5′ side of a key functional domain, the 2-oxoglutarate-Fe(II)-dependent oxygenase domain (4690 to 5160 bp); the locations of the 2 sgRNA were 4450 to 4469 bp and 4501 to 4520 bp, respectively. Similarly, all of the embryos carried mutations in TET1 (7/7), 2 homozygous and 5 biallelic mutations. In addition, TET1 proteins were not detected in 11 of 16 blastocysts, confirmed by immunocytochemistry. In this study, we successfully generated embryos lacking TET1 by introducing designed CRISPR/Cas9 system during embryogenesis. Presence of TET1 from the first injection experiment suggests that the presumable translation initiation site is not accurate. Discrepancy between genotyping and immunocytochemistry results from the second injection experiment indicates that embryos possessing TET1 protein probably have mutations in triplets, thus no premature stop codon was synthesised. Further studies will focus on identifying the role of TET1 in maintaining pluripotency and epigenetic modification during pre-implantation stage using these embryos.

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Molecular genetic study in two patients with congenital hypoaldosteronism (types I and II) in relation to previously published hormonal studies

Acta Endocrinologica ◽

10.1530/eje.0.1390096 ◽

1998 ◽

pp. 96-100 ◽

Cited By ~ 14

Author(s):

M Peter ◽

K Bunger ◽

SL Drop ◽

WG Sippell

Keyword(s):

Genetic Study ◽

Stop Codon ◽

Molecular Genetic ◽

Premature Stop Codon ◽

Type I ◽

Type Ii ◽

Loss Of Function ◽

Molecular Genetic Study ◽

Base Exchange ◽

Deficiency Type

We performed a molecular genetic study in two patients with congenital hypoaldosteronism. An original study of these patients was published in this Journal in 1982. Both index cases, a girl (patient 1) and a boy (patient 2). presented with salt-wasting and failure to thrive in the neonatal period. Parents of patient 1 were not related, whereas the parents of patient 2 were cousins. Endocrine studies had shown a defect in 18-oxidation of 18-OH-corticosterone in patient 1 and a defect in the 18-hydroxylation of corticosterone in patient 2. Plasma aldosterone was decreased in both patients, whereas 18-OH-corticosterone was elevated in patient 1 and decreased in patient 2. Plasma corticosterone and 11-deoxycorticosterone were elevated in both patients, whereas cortisol and its precursors were in the normal range. According to the nomenclature proposed by Ulick, the defects are termed corticosterone methyl oxidase (CMO) deficiency type II in patient 1, and type I in patient 2 respectively. Genetic defects in the gene CYP11B2 encoding aldosterone synthase have been described in a few cases. In patient 1, we identified only one heterozygous amino acid substitution (V386A) in exon 7, which has no deleterious effect on the enzyme activity. In patient 2 and his older brother, we identified a homozygous single base exchange (G to T) in codon 255 (GAG), causing a premature stop codon E255X (TAG). The mutant enzyme has lost the five terminal exons containing the haem binding site, and is thus a loss of function enzyme. This is only the second report of a patient with CMO deficiency type II without a mutation in the exons and exon-intron boundaries, whereas the biochemical phenotype of the two brothers with CMO deficiency type I can be explained by the patient's genotype.

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A pseudogene of caffeic acid-o-methyltransferase (COMT) in Acacia mangium: Comparative analysis with other COMT plant promoters

10.1101/269654 ◽

2018 ◽

Author(s):

Azmah Abdul Latif ◽

Sheh May Tam ◽

Wickneswari Ratnam

Keyword(s):

Stop Codon ◽

Regulatory Region ◽

Premature Stop Codon ◽

Acacia Mangium ◽

Lignin Biosynthesis ◽

Regulatory Elements ◽

Wood Products ◽

Acacia Auriculiformis ◽

Myb Transcription Factor ◽

Stem Loop

ABSTRACTAcacia mangium is a prominent tree species in the forest plantation industry of Southeast Asia, grown mainly to produce pulp and paper, and to a lesser extent wood chips and solid wood products. Lignin, a natural complex polymer used by plants for structural support and defence, has to be chemically removed during the production of quality paper. Delignification is very expensive and moreover, is an environmental pollutant. Understanding the complex mechanisms that underlie the regulation of lignin biosynthetic genes requires in-depth knowledge of not only the genes involved but also their regulatory elements. Using Thermal Asymmetric Interlaced PCR, a 770 bp promoter sequence with 93% identity with COMT1 gene from Acacia auriculiformis × A. mangium hybrid was isolated from A. mangium. Bioinformatics analysis revealed the presence of cis acting elements commonly found in other lignin biosynthesis genes such as TATA box, CAAT box, W box, AC-I and AC-11 elements. However, a nonsense mutation that created a premature stop codon was found on the first exon. Modelling of MYB transcription factor binding site on this newly isolated pseudogene shows it has binding sites for important transcription factors involved in lignin biosynthesis both in Arabidopsis thaliana and Eucalyptus grandis. Given the remarkable structures of its regulatory region, the possible structure of its transcript was detected using Mfold. Results show the transcript are capable of forming stem loop structures, a characteristic commonly attributed to presence of miRNA. Possible functions of pseudoAmCOMT1 were discussed.

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Adrenocorticotropin-Dependent Precocious Puberty of Testicular Origin in a Boy with X-Linked Adrenal Hypoplasia Congenita Due to a Novel Mutation in the DAX1 Gene

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.86.9.7816 ◽

2001 ◽

Vol 86 (9) ◽

pp. 4068-4071 ◽

Cited By ~ 31

Author(s):

Sorahia Domenice ◽

Ana Claudia Latronico ◽

Vinicius Nahime Brito ◽

Ivo Jorge Prado Arnhold ◽

Fernando Kok ◽

...

Keyword(s):

Adrenal Insufficiency ◽

Precocious Puberty ◽

Stop Codon ◽

Novel Mutation ◽

Direct Sequencing ◽

Receptor Gene ◽

Premature Stop Codon ◽

Rare Condition ◽

Coding Region ◽

Primary Adrenal Insufficiency

Primary adrenal insufficiency is a rare condition in pediatric age, and its association with precocious sexual development is very uncommon. We report a 2-yr-old Brazilian boy with DAX1 gene mutation whose first clinical manifestation was isosexual gonadotropin-independent precocious puberty. He presented with pubic hair, enlarged penis and testes, and advanced bone age. T levels were elevated, whereas basal and GnRH-stimulated LH levels were compatible with a prepubertal pattern. Chronic GnRH agonist therapy did not reduce T levels, supporting the diagnosis of gonadotropin-independent precocious puberty. Testotoxicosis was ruled out after normal sequencing of exon 11 of the LH receptor gene. At age 3 yr he developed clinical and hormonal features of severe primary adrenal insufficiency. The entire coding region of the DAX1 gene was analyzed through direct sequencing. A nucleotide G insertion between nucleotides 430 and 431 in exon 1, resulting in a novel frameshift mutation and a premature stop codon at position 71 of DAX-1, was identified. Surprisingly, steroid replacement therapy induced a clear decrease in testicular size and T levels to the prepubertal range. These findings suggest that chronic excessive ACTH levels resulting from adrenal insufficiency may stimulate Leydig cells and lead to gonadotropin-independent precocious puberty in some boys with DAX1 gene mutations.

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Resistance to Infection by Subgroups B, D, and E Avian Sarcoma and Leukosis Viruses Is Explained by a Premature Stop Codon within a Resistance Allele of the tvb Receptor Gene

Journal of Virology ◽

10.1128/jvi.76.15.7918-7921.2002 ◽

2002 ◽

Vol 76 (15) ◽

pp. 7918-7921 ◽

Cited By ~ 33

Author(s):

Sara Klucking ◽

Heather B. Adkins ◽

John A. T. Young

Keyword(s):

Stop Codon ◽

Receptor Gene ◽

Premature Stop Codon ◽

Resistance Allele ◽

Truncated Protein ◽

Single Base ◽

Single Base Pair ◽

Resistance To Infection ◽

Avian Sarcoma

ABSTRACT Here we present the first molecular characterization of the defect associated with an avian sarcoma and leukosis virus (ASLV) receptor resistance allele, tvb r. We show that resistance to infection by subgroups B, D, and E ASLV is explained by the presence of a single base pair mutation that distinguishes this allele from tvb s1, an allele which encodes a receptor for all three viral subgroups. This mutation generates an in-frame stop codon that is predicted to lead to the production of a severely truncated protein.

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Complete Androgen Insensitivity Caused by a New Frameshift Deletion of Two Base Pairs in Exon 1 of the Human Androgen Receptor Gene1

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jcem.84.5.5664 ◽

1999 ◽

Vol 84 (5) ◽

pp. 1751-1753 ◽

Cited By ~ 1

Author(s):

Brigitta Thiele ◽

Wolfgang Weidemann ◽

Doris Schnabel ◽

Gabriela Romalo ◽

Hans-Udo Schweikert ◽

...

Keyword(s):

Androgen Receptor ◽

Stop Codon ◽

Novel Mutation ◽

Reductase Activity ◽

Receptor Gene ◽

Premature Stop Codon ◽

Androgen Receptor Gene ◽

Coding Region ◽

Androgen Insensitivity ◽

Exon 1

We describe a novel mutation in exon 1 of the androgen receptor gene in a patient with complete androgen insensitivity (CAIS). Endocrine findings were typical for androgen insensitivity (testosterone serum levels in the upper limit of normal males and increased LH serum concentrations). Biochemical investigations in cultured genital skin fibroblasts of the patient showed a normal 5α-reductase activity but a complete absence of androgen binding. Western blot analysis revealed no detectable protein product. Sequence analysis of the entire coding region of the androgen receptor gene resulted in the identification of a 2-bp deletion in codon 472, causing frameshift and introduction of a premature stop codon 27 codons downstream of the mutation.

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