Premature Translational Stop Codon Research Articles

Characterization of the Transcription Termination Factor Responsible for Polarity in Archaea

The absence of a nuclear compartment permits prokaryotic ribosomes to bind nascent RNAs to initiate translation before a transcript is completely synthesized. Transcription and translation are normally tightly coupled in prokaryotes (Archaea and Bacteria) and uncoupling translation from transcription serves as a regulatory signal, permitting premature transcription termination when translation fails or is inhibited. The resultant decrease in downstream gene expression, termed polarity, is a major regulatory pathway to ensure coordinated gene expression. While the importance and widespread use of polar repression is well documented, to date, the factor responsible for polarity in archaea cells has not been explicitly identified.In bacteria, the transcription termination factor rho is responsible for polarity, but there are no rho homologues encoded in archaeal genomes. FttA (Factor that terminates transcription in Archaea), a recently identified archaeal termination factor, appears to potentially fulfill the requirements to direct polarity in archaea, but other factors may be responsible. Our primary objective, employing an unbiased genetic selection, is to identify the transcription termination factor(s) responsible for polar repression in Archaea.We have established and will present our findings resultant from a three‐tiered genetic selection for spontaneous mutations that impact the efficiency of polar repression in archaeal cells. The selections were carried out in specialized strains of Thermococcus kodakarensis, a marine, hyperthermophilic archaeon with a facile genetic system and robust laboratory growth.Briefly, genomic modifications were introduced to place a non‐essential gene with a premature translation stop codon directly upstream and within the same operon of a reporter or essential (selection) gene. In wide‐type strains, the early stop codon permits a polarity factor to recognize uncoupling of transcription and translation and terminate transcription before productive expression of the downstream essential gene. However, when the activity of a polarity factor(s) is hindered, these selective genes can be expressed at high levels. For selection, the final genes of the tryptophan and histidine synthesis pathway were chosen, constructing auxotrophic mutant strains which can only develop in the absence of tryptophan and histidine if there is some disruption in polarity‐ allowing them to become prototrophic. The spontaneous mutations resultant in reduced polar repression are easily identified through whole‐genome sequencing and we present evidence for roles of multiple factors in mediating polarity in archaeal cells.

The phenotypic landscape of a Tbc1d24 mutant mouse includes convulsive seizures resembling human early infantile epileptic encephalopathy.

Epilepsy, deafness, onychodystrophy, osteodystrophy and intellectual disability are associated with a spectrum of mutations of human TBC1D24. The mechanisms underlying TBC1D24-associated disorders and the functions of TBC1D24 are not well understood. Using CRISPR-Cas9 genome editing, we engineered a mouse with a premature translation stop codon equivalent to human S324Tfs*3, a recessive mutation of TBC1D24 associated with early infantile epileptic encephalopathy (EIEE). Homozygous S324Tfs*3 mice have normal auditory and vestibular functions but show an abrupt onset of spontaneous seizures at postnatal day 15 recapitulating human EIEE. The S324Tfs*3 variant is located in an alternatively spliced micro-exon encoding six perfectly conserved amino acids incorporated postnatally into TBC1D24 protein due to a micro-exon utilization switch. During embryonic and early postnatal development, S324Tfs*3 homozygotes produce predominantly the shorter wild-type TBC1D24 protein isoform that omits the micro-exon. S324Tfs*3 homozygotes show an abrupt onset of seizures at P15 that correlates with a developmental switch to utilization of the micro-exon. A mouse deficient for alternative splice factor SRRM3 impairs incorporation of the Tbc1d24 micro-exon. Wild-type Tbc1d24 mRNA is abundantly expressed in the hippocampus using RNAscope in situ hybridization. Immunogold electron microscopy using a TBC1D24-specific antibody revealed that TBC1D24 is associated with clathrin-coated vesicles and synapses of hippocampal neurons, suggesting a crucial role of TBC1D24 in vesicle trafficking important for neuronal signal transmission. This is the first characterization of a mouse model of human TBC1D24-associated EIEE that can now be used to screen for antiepileptogenic drugs ameliorating TBCID24 seizure disorders.

Platelet Functional and Morphological Abnormalities In a Case Of Arthrogryposis, Renal Dysfunction and Cholestasis (ARC) Syndrome With a Novel Genetic Profile

ARC syndrome is a severe autosomal recessive multisystem disorder characterized by arthrogryposis, neonatal cholestatic jaundice, renal tubular acidosis, failure to thrive, dysmorphic features, congenital heart disease, cerebral malformation, hypotonia, recurrent febrile illness and a bleeding disorder. It is associated with consanguinity and most patients do not survive beyond the first year of life. ARC syndrome is linked to loss of function in either VPS33B or VIPAS39, respectively encoding VPS33B and VPS16B, which we have shown to be interaction partners within a multiprotein complex involved in intracellular vesicular trafficking (Urban et al, Blood. 2012 Dec 13;120(25):5032-40). Earlier we determined that bleeding problems in ARC syndrome patients (where platelet aggregation abnormalities have been observed) arise from abnormal function of normally abundant platelets lacking α-granules (Lo et al, Blood. 2005;106(13):4159-66). Detailed studies of ARC syndrome platelets have been hampered by limited availability.A term 4-month old boy was observed to have significant epistaxis and petechiae requiring multiple transfusions of plasma, packed red blood cells, tranexamic acid, vitamin K and nasal packing twice, with final success over control of bleeding. INR was 1.8 and PTT was 40 with a normal platelet count of 319 during this episode. Intermittent episodes of bleeding continued throughout his course, with difficulty in stopping the bleeding. The patient was found to have jaundice with scleral icterus, cholestasis and giant cell hepatitis seen on liver biopsy. A metabolic work-up revealed significant aminoaciduria and proximal tubular acidosis. He also had failure to thrive, feeding intolerance, severe bilateral sensorineural hearing loss, limb contractures, right rocker bottom foot and clubbed left foot. MRI of the brain showed abnormal corpus callosum with non-visualization of the anterior commissure. On abdominal ultrasound, the liver was enlarged and mildly echogenic with no evidence of portal hypertension and patent vessels. Echogenic kidneys were present with nephrocalcinosis. He was referred for evaluation and possible liver transplantation.Blood film examination revealed pale-appearing platelets and transmission electron microscopy confirmed the complete absence of α-granules, making a diagnosis of ARC syndrome highly probable. Immunofluorescence confocal microscopy detected abnormalities in patient platelets with regards to: 1) size (larger than normal); 2) morphology of the tubulin ring cytoskeleton (loosely organized compared to normal); and 3) intracellular distribution of CD63 (more abundant and widely dispersed than normal). Sequencing of patient DNA detected normal VIPAS39 and apparent homozygosity for a single base pair deletion (c.1531delT, p.Tyr511ThrfsStop10) in exon 20 of VPS33B, predicted to cause a frame shift and premature translation stop codon. Gene sequencing of the parents detected the identical VPS33B mutation in the mother heterozygous to a normal allele; no mutation was found in the father. Array comparative genomic hybridization (aCGH) – which allows detection of deletions and amplification mutations of one or more exons within a given gene – uncovered an approximately 140 kb deletion mutation encompassing the entire VPS33B gene in our patient (g.91,478,004 – proximal to g.91,618.247 – distal), confirming compound heterozygosity. According to the online locus-specific ARC database (https://grenada.lumc.nl/LOVD2/ARC), this is the first association of this mutation combination with ARC syndrome.Unusually for ARC syndrome, bleeding indicative of platelet dysfunction was a major presenting symptom in this patient. While the genetic profile is unusual, the pathological platelet hallmarks of ARC syndrome were present: hypogranular platelets on blood film and absence of α-granules via electron microscopy. We were also able to detect other platelet abnormalities via immunofluorescence microscopy. These observations lead us to suspect that ARC syndrome may represent the most severe class within a possible spectrum of platelet function disorders associated with mutations in VPS33B and/or VIPAS39, a possibility that requires further study. Disclosures:No relevant conflicts of interest to declare.

First functional analysis of a novel splicing mutation in the B3GALTL gene by an ex vivo approach in Tunisian patients with typical Peters plus syndrome

Peters plus syndrome is a rare recessive autosomal disorder comprising ocular anterior segment dysgenesis, short stature, hand abnormalities and distinctive facial features. It was related only to mutations in the B3GALTL gene in the 13q12.3 region. In this study, we undertook the first functional analysis of a novel c.597-2 A>G splicing mutation within the B3GALTL gene using an ex-vivo approach. The results showed a complete skipping of exon 8 in the B3GALTL cDNA, which altered the open reading frame of the mutant transcript and generated a PTC within exon 9. This finding potentially elicits the nonsense mRNA to degradation by NMD (nonsense-mediated mRNA decay). The theoretical consequences of splice site mutations, predicted with the bioinformatics tool Human Splice Finder, were investigated and evaluated in relation to ex-vivo results. The findings confirmed the key role played by the B3GALTL gene in typical Peters-plus syndromes and the utility of mRNA analysis to understand the primary impacts of this mutation and the phenotype of the disease.

Intron-derived aberrant splicing of A20 transcript in rheumatoid arthritis

Aberrant splicing is one of the most significant components generating functional diversity in many pathological conditions. The objective of this study was to analyse the mutations or aberrant splicing of A20 transcript, the region encompassing the ovarian tumour (OTU) domain [which is functionally important as an inhibitor of nuclear factor (NF)-κB activation] in fibroblast-like synoviocytes (FLSs) from RA patients. Alterations in A20 transcripts were determined through sequence analysis of 10 clones of A20 cDNA in FLSs from each of the five RA patients. The levels of aberrant A20 transcript were measured by quantitative real-time RT-PCR with primers to specifically recognize the inserted introns. The functional role of A20 and its aberrant variants were examined by analysing NF-κB luciferase reporter activity and NF-κB-dependent target gene expression. In RA FLSs, we discovered four novel aberrant A20 transcripts, most of which resulted from insertion of partial intron 2, intron 4 and/or deletion of exon 4. In each of these FLSs, sequence analysis revealed that these aberrant insertional sequences were flanked by consensus splice donor and acceptor sequences without nucleotide substitution, suggesting alternative splicing as the likely mutational mechanism. These variants elicited a codon frame shift by creating a premature translational stop codon, and eventually, disruption of the OTU domain (which is functionally important as an inhibitor of NF-κB activation) of A20. The expression level of aberrant A20 transcript was correlated well with persisitently enhanced status of NF-κB signalling, as evident by the phosphorylation of inhibitor of NF-κB (IκB)-α and transcription of NF-κB target genes. The results suggest that A20 inactivation by the novel aberrant splicing may contribute to RA progression by inducing persistent NF-κB activation.

An exonic insertion within Tex14 gene causes spermatogenic arrest in pigs

BackgroundMale infertility is an increasing problem in all domestic species including man. Localization and identification of genes involved in defects causing male infertility provide valuable information of specific events in sperm development. Sperm development is a complex process, where diploid spermatogonia develop into haploid, highly specialized spermatozoa. Correct expression and function of various genes and their protein products are required for production of fertile sperm. We have identified an infertility defect in Finnish Yorkshire boars caused by spermatogenic arrest. The aim of this study was to locate the disease associated region using genome wide screen with the PorcineSNP60 Beadchip and identify the causal mutation by candidate gene approach.ResultsIn the Finnish Yorkshire pig population the spermatogenic arrest (SA) defect appears to be of genetic origin and causes severe degeneration of germ cells and total absence of spermatozoa. Genome wide scan with the PorcineSNP60 Beadchip localized the SA defect to porcine chromosome 12 in a 2 Mbp region. Sequencing of a candidate gene Tex14 revealed a 51 bp insertion within exon 27, which caused differential splicing of the exon and created a premature translation stop codon. The expression of Tex14 was markedly down regulated in the testis of a SA affected boar compared to control boars and no protein product was identified by Western blotting. The SA insertion sequence was also found within intron 27 in all analyzed animals, thus the insertion appears to be a possible duplication event.ConclusionIn this study we report the identification of a causal mutation for infertility caused by spermatogenic arrest at an early meiotic phase. Our results highlight the role of TEX14 specifically in spermatogenesis and the importance of specific genomic remodeling events as causes for inherited defects.

Genetic analysis of functional redundancy of BRM ATPase and ATSWI3C subunits of Arabidopsis SWI/SNF chromatin remodelling complexes

In yeast and mammals, ATP-dependent chromatin remodelling complexes of the SWI/SNF family play critical roles in the regulation of transcription, cell proliferation, differentiation and development. Homologues of conserved subunits of SWI/SNF-type complexes, including Snf2-type ATPases and SWI3-type proteins, participate in analogous processes in Arabidopsis. Recent studies indicate a remarkable similarity between phenotypic effects of mutations in the SWI3 homologue ATSWI3C and bromodomain-ATPase BRM genes. To verify the extent of functional similarity between BRM and ATSWI3C, we have constructed atswi3c brm double mutants and compared their phenotypic traits to those of simultaneously grown single atswi3c and brm mutants. In addition to inheritance of characteristic developmental abnormalities shared by atswi3c and brm mutants, some additive brm-specific traits were also observed in the atswi3c brm double mutants. Unlike atswi3c, the brm mutation results in the enhancement of abnormal carpel development and pollen abortion leading to complete male sterility. Despite the overall similarity of brm and atswi3c phenotypes, a critical requirement for BRM in the differentiation of reproductive organs suggests that its regulatory functions do not entirely overlap those of ATSWI3C. The detection of two different transcript isoforms indicates that BRM is regulated by alternative splicing that creates an in-frame premature translation stop codon in its SNF2-like ATPase coding domain. The analysis of Arabidopsis mutants in nonsense-mediated decay suggests an involvement of this pathway in the control of alternative BRM transcript level.

Mutations in phospholipase C epsilon 1 are not sufficient to cause diffuse mesangial sclerosis

Diffuse mesangial sclerosis occurs as an isolated abnormality or as a part of a syndrome. Recently, mutations in phospholipase C epsilon 1 (PLCE1) were found to cause a nonsyndromic, autosomal recessive form of this disease. Here we describe three children from one consanguineous kindred of Pakistani origin with diffuse mesangial sclerosis who presented with congenital or infantile nephrotic syndrome. Homozygous mutations in PLCE1 (also known as KIAA1516, PLCE, or NPHS3) were identified following genome-wide mapping of single-nucleotide polymorphisms. All affected children were homozygous for a four-basepair deletion in exon 3, which created a premature translational stop codon. Analysis of the asymptomatic father of two of the children revealed that he was also homozygous for the same mutation. We conclude this nonpenetrance may be due to compensatory mutations at a second locus and that mutation within PLCE1 is not always sufficient to cause diffuse mesangial sclerosis.

An Intron-Derived Insertion/Truncation Mutation in the BCR-ABL Kinase Domain in Chronic Myeloid Leukemia Patients Undergoing Kinase Inhibitor Therapy

Although targeted inhibition of BCR-ABL with imatinib is an effective therapy for patients with chronic myeloid leukemia (CML), a minority of patients acquire mutations in the BCR-ABL kinase domain, resulting in imatinib resistance. The spectrum of kinase domain mutations discovered to date is quite heterogeneous, consisting almost exclusively of single nucleotide substitutions affecting key amino acids that regulate drug binding or BCR-ABL function. Here, we describe an alternative kinase domain insertion/truncation mutation in three CML patients undergoing kinase inhibitor therapy. In each of these patients, direct DNA sequencing of BCR-ABL RT-PCR products revealed that the same 35 nucleotides from ABL intron 8 had been inserted at the normal exon 8 to 9 splice junction. This 35-bp intronic sequence was flanked by excellent consensus splice donor and acceptor sequences, suggesting alternative splicing as the likely mutational mechanism. The insertion created a premature translational stop codon after 10 intron-encoded amino acids (amino acid 484). This resulted in truncation of 653 C-terminal amino acids, which included part of the kinase domain and the entire "last exon" region. These findings demonstrate that kinase domain insertions are an alternative (and not entirely uncommon) mutational mechanism in CML patients undergoing kinase inhibitor therapy.

An Intron-Derived Insertion/Truncation Mutation in the BCR-ABL Kinase Domain in Three CML Patients Undergoing Kinase Inhibitor Therapy.

Although targeted inhibition of BCR-ABL with imatinib is an effective therapy for patients with chronic myeloid leukemia, a minority acquire mutations in the kinase domain (KD) that cause imatinib resistance. The spectrum of KD mutations thus far discovered, although quite heterogeneous, includes almost exclusively single nucleotide substitutions in key amino acids regulating drug binding or BCR-ABL function. Here, we describe a KD insertion/truncation mutation in 3 CML patients undergoing kinase inhibitor therapy. Two of these patients were being treated with imatinib (for 12 and 17 months), and one with dasatinib (for 13 months after a prior relapse while on imatinib). Suspected drug resistance was assessed by direct DNA sequencing of a BCR-ABL PCR product extending to the end of the kinase domain. Each of these 3 patients had 35 nucleotides from ABL intron 8 inserted at the normal exon 8–9 splice junction, after nucleotide 1423 (amino acid 475) of Genbank cDNA clone NM_005157. In all 3 cases, the mutation was co-expressed with wild type BCR-ABL sequence. The inserted sequence is derived from intron 8, beginning 1151 bp downstream from the normal splice donor site at the end of exon 8. This 35 bp intronic sequence is flanked by excellent consensus splice donor and acceptor sequences, suggesting alternative splicing as the likely mutational mechanism. The insertion creates a premature translational stop codon after 10 intron-encoded amino acids (figure), thus truncating 653 C-terminal amino acids including part of the KD and the entire last exon region - including a proline-rich domain, 3 nuclear localization signals, a DNA-binding domain, an actin-binding domain, and a nuclear export signal. These 3 insertion mutation cases were detected in our diagnostic clinical molecular pathology laboratory after sequencing 174 cases referred to us for suspected kinase inhibitor resistance, 78 of which contained a detectable mutation. The estimated prevalence of the exon 8/9 insertion/truncation mutation is then approximately 1.7% among patients with suspected drug resistance, and this mutation constitutes approximately 3.8% of all mutations.Conclusion: Kinase domain insertions are an alternative (and not entirely uncommon) mutational mechanism in CML patients undergoing kinase inhibitor therapy. The functional significance in terms of kinase activity and drug resistance remains to be addressed. [Display omitted]

β Subunits of Voltage-gated Sodium Channels Are Novel Substrates of β-Site Amyloid Precursor Protein-cleaving Enzyme (BACE1) and γ-Secretase

Sequential processing of amyloid precursor protein (APP) by membrane-bound proteases, BACE1 and gamma-secretase, plays a crucial role in the pathogenesis of Alzheimer disease. Much has been discovered on the properties of these proteases; however, regulatory mechanisms of enzyme-substrate interaction in neurons and their involvement in pathological changes are still not fully understood. It is mainly because of the membrane-associated cleavage of these proteases and the lack of information on new substrates processed in a similar way to APP. Here, using RNA interference-mediated BACE1 knockdown, mouse embryonic fibroblasts that are deficient in either BACE1 or presenilins, and BACE1-deficient mouse brain, we show clear evidence that beta subunits of voltage-gated sodium channels are sequentially processed by BACE1 and gamma-secretase. These results may provide new insights into the underlying pathology of Alzheimer disease.

Duplicated coding sequence in the waxy allele of tropical glutinous rice ( Oryza sativa L.)

Rice starch consists of amylopectin and amylose, the latter component being controlled by the Waxy ( Wx) gene. Two alleles Wx a and Wx b were found to regulate the quantitative level of the Wx protein as well as the amylose content. A comparison of the genomic sequences of rice Wx genes responsible for the synthesis of grain amylose was made between two pairs of glutinous and non-glutinous reverse mutants. By comparing their 5′ splice codons in the first intron, the low-amylose and glutinous rices were characterized as the Wx b allele based on the G-to-T base substitution, which caused an aberrant splicing. When the entire genomic sequences of Wx b were compared, a unique duplication of 23 bp was found to be present only in the glutinous mutants. This 23 bp insertion was truly unique for tropical glutinous rice because of its similarity between the 800 bp sequences around the duplicated sequences of 24 glutinous varieties and two annual diploid wild rice accessions. This in-frame duplication created a premature translation stop codon at 78 bp downstream. The insertion was unique and none of the structure was identical to any transposon or retrotransposon ever reported in rice before. This unique duplicated sequence opens up opportunities to develop PCR-based markers useful for classifying grain amylose in rice.

Mutation of a transcription factor, TFCP2L3, causes progressive autosomal dominant hearing loss, DFNA28.

We ascertained a large American family with an autosomal dominant form of progressive non-syndromic sensorineural hearing loss. After excluding linkage to known deafness loci, we performed a genome-wide scan and found linkage to marker GAAT1A4 on chromosome 8q22 (LOD=5.12 at theta=0), and this locus was designated DFNA28. Sequencing of six candidate genes in the 1.4 cM linked region identified a frameshift mutation (1609-1610insC) resulting in a premature translation stop codon in exon 14 of the gene TFCP2L3 (transcription factor cellular promoter 2-like 3). TFCP2L3 is a member of a family of transcription factor genes whose archetype is TFCP2, a mammalian homolog of the Drosophila gene grainyhead. Northern blot analyses and in situ hybridization studies show that mouse Tfcp2l3 is expressed in many epithelial tissues, including cells lining the cochlear duct, at embryonic day 18.5 and postnatal day 5.

Regulation of the apolipoprotein B in heterozygous hypobetalipoproteinemic knock-out mice expressing truncated apoB, B81. Low production and enhanced clearance of apoB cause low levels of apoB.

Low levels of cholesterol are protective against development of coronary artery disease. Heterozygous hypobetalipoproteinemic individuals expressing truncated apolipoprotein (apo)B as a result of mutation in the apob gene have low levels of cholesterol and apoB in their plasma. To study the molecular mechanism of low levels of apoB in these individuals, we employed a previously reported knock out mouse model generated by targeted modification of the apob gene. The heterozygous, apoB-100/B-81, mice express full length and truncated apoB, B-81, and have 20 and 35% lower levels of total cholesterol and apoB, respectively, when compared to WT (apoB-100/B-100) mice. The majority of the truncated apoB, B-81, fractionated in the VLDL- density range. The mechanism of low levels of apoB in B-100/B-81 mice was examined. Total hepatic apoB mRNA levels decreased by 15%, primarily due to lower levels of apoB-81 mRNA. Since apoB mRNA transcription rates were similar in B-100/B-100 and B-100/B-81 mice, low levels of mutant apoB-81 mRNA occurred by enhanced degradation of apoB mRNA transcript containing premature translational stop codon. ApoB synthesis measured on isolated hepatocytes decreased in B-100/B-81 mice by 35%, while apoB-48, apoE, and apoAI syntheses remained unchanged. Metabolic studies using whole animal showed a 32% decrease in triglyceride secretion rates, consistent with the apoB secretion rates. Inhibition of receptor-mediated clearance of apoB-81-containing particles resulted in greater relative accumulation of apoB-81 in plasma than apoB-100, suggesting enhanced clearance of apoB-81-containing particles. These results demonstrate that low levels of apoB in heterozygous hypobetalipoproteinemic mice occurs by low rates of apoB secretion, and increased clearance of truncated apoB. Similar mechanisms appear to contribute to low levels of apoB in hypobetalipoproteinemic humans.

Absence of opioid stress-induced analgesia in mice lacking beta-endorphin by site-directed mutagenesis.

A physiological role for beta-endorphin in endogenous pain inhibition was investigated by targeted mutagenesis of the proopiomelanocortin gene in mouse embryonic stem cells. The tyrosine codon at position 179 of the proopiomelanocortin gene was converted to a premature translational stop codon. The resulting transgenic mice display no overt developmental or behavioral alterations and have a normally functioning hypothalamic-pituitary-adrenal axis. Homozygous transgenic mice with a selective deficiency of beta-endorphin exhibit normal analgesia in response to morphine, indicating the presence of functional mu-opiate receptors. However, these mice lack the opioid (naloxone reversible) analgesia induced by mild swim stress. Mutant mice also display significantly greater nonopioid analgesia in response to cold water swim stress compared with controls and display paradoxical naloxone-induced analgesia. These changes may reflect compensatory upregulation of alternative pain inhibitory mechanisms.

Differential and tissue-specific expression of a gene family for tyrosine/dopa decarboxylase in opium poppy.

Two early and potential rate-limiting steps in the biosynthesis of isoquinoline alkaloids, such as morphine and codeine, in opium poppy (Papaver somniferum) involve decarboxylation of L-tyrosine and L-dihydroxyphenylalanine (L-dopa) to yield tyramine and dopamine, respectively. A DNA fragment was amplified by polymerase chain reaction (PCR) using degenerate primers designed to two highly conserved domains found in other aromatic amino acid decarboxylases. A poppy seedling cDNA library was screened with this PCR product and a cDNA (cTYDC1) for tyrosine/dopa decarboxylase (TYDC/DODC) was isolated. Two other independent cDNAs (cTYDC2 and cTYDC3) encoding TYDC/DODC were isolated by heterologous screening with a plant tryptophan decarboxylase (TDC) cDNA as probe. A poppy genomic library was screened with cTYDC1 and two intronless genomic clones (gTYDC1 and gTYDC4) were isolated. The deduced amino acid sequences of all poppy clones share extensive identity with other reported pyridoxal phosphate-dependent decarboxylases from both plants and animals. Based on sequence homology, members of the gene family were divided into two subsets (cTYDC1 and gTYDC4; cTYDC2 and cTYDC3) of proteins with predicted M(r) = 56,983 and 59,323, respectively. Within each subset the clones exhibit greater than 90% identity, whereas clones between subsets share less than 75% identity. Expression of gTYDC1 and cTYDC2 as beta-galactosidase fusion proteins in Escherichia coli resulted in catalytically active enzymes immunodetectable with TDC-specific polyclonal antibodies. Each enzyme showed marginally higher substrate specificity for L-dopa over L-tyrosine, but did not accept L-tryptophan and L-phenylalanine as substrates. Genomic DNA blot-hybridization analysis revealed 6 to 8 genes homologous to cTYDC1 and 4 to 6 genes homologous to cTYDC2 in the tetraploid poppy genome. A premature translation stop codon was found in the gTYDC4 clone suggesting that it may not encode a functional protein. RNA blot-hybridization with probes specific to the gTYDC1- or cTYDC2-like subsets showed that members of the TYDC gene family are differentially expressed in various plant tissues.

Characterization of single base substitutions In edited apolipoprotein B transcripts

Mature RNA transcripts from a single eukaryotic gene may contain different nucleotide sequences, ranging from alternately spliced exons to transcripts from separate alleles differing by only one base. Our laboratory and others have recently reported another class of RNA sequence differences, occurring in transcripts from the single copy apolipoprotein B (apoB) gene. A unique RNA editing mechanism allows expression of the CAA glutamine codon encoded by the apoB gene at nucleotide 6666, or terminates translation by the introduction of a premature UAA translational stop codon. In this study, we used the polymerase chain reaction (PCR) to amplify and characterize edited apoB RNA transcripts differing by a single nucleotide. Amplification and sequence analysis from small quantities of total RNA will facilitate the study of RNA editing and transcription in general.

Human apolipoprotein B (apoB) mRNA: identification of two distinct apoB mRNAs, an mRNA with the apoB-100 sequence and an apoB mRNA containing a premature in-frame translational stop codon, in both liver and intestine.

Human apolipoprotein B (apoB) is present in plasma as two separate isoproteins, designated apoB-100 (512 kDa) and apoB-48 (250 kDa). ApoB is encoded by a single gene on chromosome 2, and a single nuclear mRNA is edited and processed into two separate apoB mRNAs. A 14.1-kilobase apoB mRNA codes for apoB-100, and the second mRNA, which codes for apoB-48, contains a premature stop codon generated by a single base substitution of cytosine to uracil at nucleotide 6538, which converts the translated CAA codon coding for the amino acid glutamine at residue 2153 in apoB-100 to a premature in-frame stop codon (UAA). Two 30-base synthetic oligonucleotides (nucleotides 6523-6552 of apoB mRNA), designated apoB-Stop and apoB-Gln, were synthesized containing the complementary sequence to the stop codon (UAA) and glutamine codon (CAA), respectively. Analysis of intestinal apoB mRNA by hybridization with apoB-Stop and apoB-Gln probes and sequence analysis of apoB clones in two independent human small intestinal cDNA libraries established that intestinal apoB mRNA contained both the apoB mRNA that codes for apoB-100 and the apoB mRNA containing the premature in-frame stop codon, which codes for apoB-48. Investigation of hepatic apoB mRNA and two hepatic cDNA libraries by hybridization with the apoB-Stop and apoB-Gln synthetic probes as well as by cDNA sequencing revealed that liver apoB mRNA also contains both the apoB-100 mRNA and the apoB-48 mRNA containing the stop codon. The combined results from these studies establish that both human intestine and liver contain the two distinct apoB mRNAs, an mRNA that codes for apoB-100 and an apoB mRNA that contains the premature stop codon, which codes for apoB-48. The premature in-frame stop codon is not tissue specific and is present in both human liver and intestine.