ATG Initiation Codon Research Articles

Molecular Identification and Characterization of a Family of Kinases with Homology to Ca2+/Calmodulin-dependent Protein Kinases I/IV

Despite the critical importance of Ca(2+)/calmodulin (CaM)-dependent protein kinase (CaMK) II signaling in neuroplasticity, only a limited amount of work has so far been available regarding the presence and significance of another predominant CaMK subfamily, the CaMKI/CaMKIV family, in the central nervous system. We here searched for kinases with a core catalytic structure similar to CaMKI and CaMKIV. We isolated full-length cDNAs encoding three mouse CaMKI/CaMKIV-related kinases, CLICK-I (CL1)/doublecortin and CaM kinase-Like (DCAMKL)1, CLICK-II (CL2)/DCAMKL2, and CLICK-I,II-related (CLr)/DCAMKL3, the kinase domains of which had an intermediate homology not only to CaMKI/CaMKIV but also to CaMKII. Furthermore, CL1, CL2, and CLr were highly expressed in the central nervous system, in a neuron-specific fashion. CL1alpha and CL1beta were shorter isoforms of DCAMKL1, which lacked the doublecortin-like domain (Dx). In contrast, CL2alpha and CL2beta contained a full N-terminal Dx, whereas CLr only possessed a partial and dysfunctional Dx. Interestingly, despite a large similarity in the kinase domain, CL1/CL2/CLr had an impact on CRE-dependent gene expression distinct from that of the related CaMKI/CaMKIV and CaMKII. Although these were previously shown to activate Ca(2+)/cAMP-response element-binding protein (CREB)-dependent transcription, we here show that CL1 and CL2 were unable to significantly phosphorylate CREB Ser-133 and rather inhibited CRE-dependent gene expression by a dominant mechanism that bypassed CREB and was mediated by phosphorylated TORC2.

Novel Functional Single Nucleotide Polymorphisms in the Latent Transforming Growth Factor-β Binding Protein-1L Promoter: Effect on Latent Transforming Growth Factor-β Binding Protein-1L Expression Level and Possible Prognostic Significance in Ovarian Cancer

Latent transforming growth factor (TGF)binding proteins (LTBPs) play important roles in the secretion and activation of TGF. We previously reported that LTBP-1L is overexpressed in some patients with ovarian cancer. To clarify the molecular mechanism of LTBP-1L regulation, we analyzed DNA sequences in the promoter region of LTBP-1L and identified two novel single nucleotide polymorphisms, 202G/C and 20A/C. While the alleles with 202C and 20C were initially reported, our data demonstrated that 202G and 20A are common in both ovarian cancer patients and healthy patients in the Japanese population. Luciferase reporter assays revealed that the G-A haplotype induced transcriptional activation in a Sp1-dependent manner. Electrophoretic mobility shift assays showed that increased binding affinity of Sp1 to the promoter with 202G and 20A. Interestingly , ovarian cancer patients (n 42) with G-A/G-A homozygous genotype had increased expression of LTBP-1 and apparently poorer survival than those with other genotypes (P 0.02). These findings suggest that the single nucleotide polymorphisms 202G/C and 20A/C on the LTBP-1L promoter may affect the clinical outcome of ovarian cancer patients , probably via up-regulating protein expression. Further studies using a larger number of samples will definitively determine the correlation between LTBP-1 haplotype and clinical behavior of ovarian cancer. (J Mol Diagn 2006, 8:342–350; DOI: 10.2353/jmoldx.2006.050133) Transforming growth factor (TGF)is a potent growth inhibitor for most cell types, including epithelial cells. The secretion of TGFby tumor cells may contribute to tumor suppression by autocrine growth inhibition, but on the other hand, it may also promote tumor progression by stimulating tumor invasion and angiogenesis, and inhibiting immune response. TGFis synthesized as latent high-molecular weight complexes, composed of TGF, the NH2-terminal part of the TGFprecursor, and the latent TGFbinding protein (LTBP). LTBP is a glycoprotein with a molecular weight of more than 190 kd; it possesses 16 to 18 epidermal growth factor (EGF)-like domains and several repeats of a unique motif containing eight cysteine residues. Four isoforms of LTBP have been found in mammalian species (LTBP-1 to LTBP4). LTBP-1 binds to TGF1 through one of the eight cysteine motifs and facilitates assembly and secretion and activation of TGF1. Immunoelectronmicroscopic observations indicate that LTBP-1 is one of the extracellular microfibrillar components; it targets TGF1 to extracellular structures and participates in the activation of latent TGF1, perhaps by concentrating latent TGF1 on the cell surface where activation occurs. LTBP-1S (short) and LTBP-1L (long) are derived from independent promoters and alternative splicing between codons 145 and 146 of LTBP-1S. LTBP-1L has a N-terminal extension of 346 amino acids that is not found in the LTBP-1S. The N-terminal extension contains an EGF-like domain that facilitates matrix incorporation, and LTBP-1L has been confirmed to associate more efficiently than LTBP-1S with the extracellular matrix.

A Variation of the Translation Attenuation Model Can Explain the Inducible Regulation of the pBC16 Tetracycline Resistance Gene in Bacillus subtilis

Expression of the tet resistance gene from plasmid pBC16 is induced by the antibiotic tetracycline, and induction is independent of the native promoter for the gene. The nucleotide sequence at the 5' end of the tet mRNA (the leader region) is predicted to assume a complex secondary structure that sequesters the ribosome binding site for the tet gene. A spontaneous, constitutively expressed tet gene variant contains a mutation predicted to provide the tet gene with a nonsequestered ribosome binding site. Lastly, comparable levels of tet mRNA can be demonstrated in tetracycline-induced and uninduced cells. These results are consistent with the idea that the pBC16 tet gene is regulated by translation attenuation, a model originally proposed to explain the inducible regulation of the cat and erm genes in gram-positive bacteria. As with inducible cat and erm genes, the pBC16 tet gene is preceded by a translated leader open reading frame consisting of a consensus ribosome binding site and an ATG initiation codon, followed by 19 sense codons and a stop codon. Mutations that block translation of cat and erm leaders prevent gene expression. In contrast, we show that mutations that block translation of the tet leader result in constitutive expression. We provide evidence that translation of the tet leader peptide coding region blocks tet expression by preventing the formation of a secondary-structure complex that would, in the absence of leader translation, expose the tet ribosome binding site. Tetracycline is proposed to induce tet by blocking or slowing leader translation. The results indicate that tet regulation is a variation of the translation attenuation model.

Evidence for a functional genetic polymorphism of the human thiosulfate sulfurtransferase (Rhodanese), a cyanide and H 2S detoxification enzyme

Rhodanese or thiosulfate sulfurtransferase (TST) is a mitochondrial matrix enzyme that plays roles in cyanide detoxification, the formation of iron–sulfur proteins and the modification of sulfur-containing enzymes. Transsulfuration reaction catalyzed by TST is also involved in H 2S detoxification. To date, no polymorphism of the human TST gene had been reported. We developed a screening strategy based on a PCR-SSCP method to search for mutations in the 3 exons of TST and their proximal flanking regions. This strategy has been applied to DNA samples from 50 unrelated French individuals of Caucasian origin. Eleven polymorphisms consisting in seven nucleotide substitutions in non-coding regions, two silent mutations and two missense mutations were characterized. The functional consequences of the identified mutations were assessed in vivo by measurement of erythrocyte TST activity and/or in vitro using heterologous expression in Saccharomyces cerevisiae or transient transfection assay in HT29 and Caco-2 cell lines. The P 285A variant appears to encode a protein with a 50% decrease of in vitro intrinsic clearance compared to the wild-type enzyme. Additionally, the six polymorphisms located upstream the ATG initiation codon are responsible for a significant decrease (ranging from 40% to 73%) in promoter activity of a reporter gene compared to the corresponding wild-type sequence. This work constitutes the first report of the existence of a functional genetic polymorphism affecting TST activity and should be of great help to investigate certain disorders for which impairment of CN - or H 2S detoxification have been suggested to be involved.

A Novel Ded1-like RNA Helicase Interacts with the Y-box Protein ctYB-1 in Nuclear mRNP Particles and in Polysomes

We have characterized a novel mRNA-binding protein, designated hrp84, in the dipteran Chironomus tentans and identified it as a DEAD-box RNA helicase. The protein contains the typical helicase core domain, a glycine-rich C-terminal part and a putative nuclear export signal in the N terminus. The protein belongs to the Ded1 subgroup of DEAD-box helicases, which is highly conserved from yeast (Ded1p) to mammals (DDX3). In tissue culture cells, hrp84 is present both in the nucleus and cytoplasm and, as shown by in vivo UV cross-linking, is bound to mRNA in both compartments. Immunoprecipitation experiments revealed that hpr84 is associated with the C. tentans homologue (ctYB-1) of the vertebrate Y-box protein YB-1 both in the nucleus and cytoplasm, and the two proteins also appear together in polysomes. The interaction is likely to be direct as shown by in vitro binding of purified components. We conclude that the mRNA-bound hrp84.ctYB-1 complex is formed in the nucleus and is translocated with mRNA into the cytoplasm and further into polysomes. As both Ded1 and YB-1 are known to regulate the initiation of translation, we propose that the RNA helicase-Y-box protein complex affects the efficiency of mRNA translation, presumably by modulating the conformation of the mRNP template.

All-Trans Retinoic Acid Induces XAF1 Expression Through an Interferon Regulatory Factor-1 Element in Colon Cancer

X-linked inhibitor of apoptosis protein (XIAP)-associated factor 1 (XAF1) is a novel tumor suppressor and interferon (IFN)-stimulated gene. All-trans retinoic acid (ATRA) exerts an antiproliferative effect on tumor cells through up-regulation of IFN regulatory factor 1 (IRF-1) and the downstream IFN-stimulated genes. The aim of this study was to determine the effect and mechanism of ATRA on XAF1 expression and the role of XAF1 in ATRA-induced growth inhibition in colon cancer. Gene expression is detected by reverse-transcription polymerase chain reaction and immunoblotting. The transcription activity of XAF1 promoter is examined by luciferase reporter assay. The activity of IFN regulatory factor binding element (IRF-E) is assessed by electrophoretic mobility shift assay and chromatin immunoprecipitation assay. Cell growth is evaluated by both in vitro and in vivo in nude mice xenografts. IFN-alfa stimulates XAF1 promoter activity in the colon cancer cells Lovo and SW1116 dose-dependently. An IRF-1 binding element (IRF-E-XAF1) is found in the -30 to -38 nucleotide region upstream of the ATG initiator codon of the XAF1 gene. Site-directed mutagenesis of IRF-E-XAF1 abrogates native and IFN-induced promoter activity and binding capacity. ATRA induces XAF1 expression both in vitro and in vivo through interaction with IRF-E-XAF1. Overexpression of XAF1 increases cell susceptibility to ATRA-induced growth suppression both in vitro and in vivo. Furthermore, the effect of ATRA on XAF1 expression is independent of the promoter methylation and the subcellular distribution of XIAP. XAF1 participates in ATRA-induced growth suppression through IRF-1-mediated transcriptional regulation.

The proximal promoter region of mTert is sufficient to regulate telomerase activity in ES cells and transgenic animals

BackgroundThe reverse transcriptase of telomerase (Tert) controls telomerase activity maintaining the end of linear chromosomes in eukaryotic cells. Telomerase function is highly active in undifferentiated multipotent stem cells, decreases with cell differentiation and is generally absent from most somatic cells in the adult. Its absence is responsible of telomeres shortening in such somatic cells. Using an in vivo transgenic model and an in vitro culture differentiation of adult stem cells, we examined the elements of the mouse Tert (mTert) promoter that control telomerase activity.ResultsThree constructs comprising 1, 2 or 5 kb of the mTert promoter sequence coupled to the coding sequence of the green fluorescent protein (EGFP) were electroporated into embryonic stem (ES) cells. Transformed ES cells were able to mimic the expected mTert expression, which was associated to green fluorescence. One and 5 kb promoter produced the higher expression of EGFP, on ES cells. When ES cells were allowed to differentiate to embryoid bodies and to other cell types, they lost gradually the expression of mTert-EGFP as consequence of differentiation. No differences were found among the three constructs analyzed. We then generated transgenic mice with the three constructs. Expression of the reporter gene was monitored by reverse transcription-PCR analysis and EGFP visualization. The mRNA expression of the three constructs was lower than the endogenous mTert, but mimicked the endogenous mTert transcription pattern; however, no fluorescent expression of EGFP was detected in adult tissues. EGFP expression of the three constructs was visualized at the blastocysts stage and in new ES cells generated from them; in the germinal ring of E13 dpc foetuses; in ES-like colonies and in germinal stem cells generated from neonatal and adult testis cells; and in neuroesferes generated from E14 dpc foetuses' brain cells.ConclusionThe 1 kb promoter upstream of the initiating ATG codon of mTert contains all the regulatory elements to control telomerase expression in ES cells during in vitro loss of pluripotency. The transgenic mouse lines generated represent an appropriate system to analyze the expression of mouse Tert gene under physiological condition and during establishment of stem cell lines generated from embryonic or adult tissues.

Nucleotide sequence of the gene encoding cis-biphenyl dihydrodiol dehydrogenase (bphB) and the expression of an active recombinant His-tagged bphB gene product from a PCB degrading bacterium, Pseudomonas putida OU83.

The nucleotide sequence of the bphB gene of Pseudomonas putida strain OU83 was determined. The bphB gene, which encodes cis-biphenyl dihydrodiol dehydrogenase (BDDH), was composed of 834 base pairs with an ATG initiation codon and a TGA termination codon. It can encode a polypeptide of 28.91 kDa, containing 277 amino acids. Promoter-like and ribosome-binding sequences were identified upstream of the bphB gene. The bphB nucleotide sequence was used to produce His-tagged BDDH, in Escherichia coli. The His-tagged BDDH construction, carrying a single 6 x His tail on the N-terminal portion, was active. The molecular mass of the native enzyme was 128 kDa and on SDS-PAGE analysis the molecular mass was 31 kDa. This enzyme requires NAD+ for its activity and its optimum pH is 8.5. Nucleotide and the deduced amino acid sequence analyses revealed a high degree of homology between the bphB gene from Pseudomonas putida OU83 and the bphB genes from P. cepacia LB400 and P. pseudoalcaligenes KF707.

Familial Parkinson Mutant α-Synuclein Causes Dopamine Neuron Dysfunction in Transgenic Caenorhabditis elegans

Mutations in alpha-synuclein gene cause familial form of Parkinson disease, and deposition of wild-type alpha-synuclein as Lewy bodies occurs as a hallmark lesion of sporadic Parkinson disease and dementia with Lewy bodies, implicating alpha-synuclein in the pathogenesis of Parkinson disease and related neurodegenerative diseases. Dopamine neurons in substantia nigra are the major site of neurodegeneration associated with alpha-synuclein deposition in Parkinson disease. Here we establish transgenic Caenorhabditis elegans (TG worms) that overexpresses wild-type or familial Parkinson mutant human alpha-synuclein in dopamine neurons. The TG worms exhibit accumulation of alpha-synuclein in the cell bodies and neurites of dopamine neurons, and EGFP labeling of dendrites is often diminished in TG worms expressing familial Parkinson disease-linked A30P or A53T mutant alpha-synuclein, without overt loss of neuronal cell bodies. Notably, TG worms expressing A30P or A53T mutant alpha-synuclein show failure in modulation of locomotory rate in response to food, which has been attributed to the function of dopamine neurons. This behavioral abnormality was accompanied by a reduction in neuronal dopamine content and was treatable by administration of dopamine. These phenotypes were not seen upon expression of beta-synuclein. The present TG worms exhibit dopamine neuron-specific dysfunction caused by accumulation of alpha-synuclein, which would be relevant to the genetic and compound screenings aiming at the elucidation of pathological cascade and therapeutic strategies for Parkinson disease.

Characterisation of the mulibrey nanism-associated TRIM37 gene: Transcription initiation, promoter region and alternative splicing

The TRIM37 gene encodes a peroxisomal protein of unknown function. Mutations in TRIM37 underlie mulibrey nanism, a rare autosomal recessively inherited disorder with severe growth failure of prenatal onset, constrictive pericardium, hepatomegaly and characteristic dysmorphic features. Eleven mulibrey nanism-associated mutations have been identified. We here characterised TRIM37 further by mapping the transcription initiation site and promoter region as well as by analysing splice variants. By primer extension analysis, several transcription initiation sites were localised to a region between − 246 and − 373 relative to the ATG codon for translation initiation. Basal promoter activity was mapped within 600 nucleotides upstream from the translation initiation site using promoter-luciferase reporter constructs. Several alternative splice variants of TRIM37 exist in databases. Most of these predict non-functional protein products, are expressed at low levels and are thus likely to be targets for nonsense-mediated mRNA decay. A novel splice variant, TRIM37b, with an alternative termination codon and 3′untranslated region (UTR) transcribed from an exon 16 kb downstream from exon 24, predicts an identical protein product with the previously identified transcript, TRIM37a. As seen by Northern blot analysis and quantitative real-time PCR, both transcripts are highly expressed in testis, whereas in other tissues TRIM37a is prominent. The 3′UTR of the PPM1E gene in the opposite strand overlaps TRIM37b. These data suggest that TRIM37 expression is regulated by several mechanisms: through nonsense surveillance of non-functional transcripts, as well as through 3′UTR regulatory sequences and/or naturally occurring antisense RNAs especially in testis.

Rapid and easy thermodynamic optimization of the 5′-end of mRNA dramatically increases the level of wild type protein expression in Escherichia coli

Low levels of expression in Escherichia coli are often observed when using wild type proteins. The addition of an N-terminal His-tag to these same proteins dramatically improves the level of expression. We therefore concluded that post-transcriptional regulation and in particular translational regulation are probably influenced by the presence of the tag. The RNAfold program was used to analyze the 5′-end of the encoding mRNA, and more precisely the area encompassing the Shine–Dalgarno region and the initiation codon ATG. We observed that hairpin loops can be formed and that the stability of these loops correlates with the level of protein expression in E. coli. Our recently developed cloning technology by PCR fragment integration allows us to easily and rapidly introduce mutations anywhere within a gene. In our studies, we used this technology to destabilize the predicted hairpin by introducing silent mutations within the first 72 nucleotides of the coding sequence. As a result of the decreased stability of the RNA hairpins, we could significantly increase the level of expression of wild type proteins and without having to rely on the use of tags in E. coli. In addition, our studies allow us to predict whether or not a protein will be expressed without additional engineering of its encoding gene.

Improvement of Recombination Efficiency by Mutation of Red Proteins

The Red recombination system of the bacteriophage lambda is a useful tool for engineering bacterial artificial chromosomes (BACs) because desired mutations can be made in any part of the DNA, even in large DNAs, independent of the presence of appropriate restriction enzyme sites. Many have tried changing the inducible promoter or decreasing the copy number of Red genes in Escherichia coli to improve the recombination efficiency. Here we describe a novel method for increasing the recombination efficiency of the Red system by mutating the Red proteins. Mutant Red alpha and Red beta independently increased recombination efficiency. We found that the 18-bp 5' untranslated region (UTR) upstream from the ATG initiation codon of Gam protein is important for recombination efficiency. Although low rates of recombination have been observed in systems using high copy number plasmids, when we performed Red recombination using a high copy number plasmid containing 5'-UTR, mutant Red alpha, and mutant Red beta, recombination increased approximately 145-fold. The novel possibility that mutant Red alpha and Red beta can increase recombination efficiency indicates a new direction for improving recombination efficiency of expression systems that use low or high copy number plasmids or a defective prophage integrated into the E. coli chromosome.

Membrane Topology of the STT3 Subunit of the Oligosaccharyl Transferase Complex

The highly conserved membrane protein STT3 is part of the oligosaccharyl transferase complex in the endoplasmic reticulum of eukaryotic cells. Various experimental observations strongly suggest that STT3 contains the active site of the complex. Here, we report a detailed topology study of STT3 from two different organisms, Saccharomyces cerevisiae and mouse, using in vivo and in vitro topology mapping assays. Our results suggest that STT3 has 11 transmembrane helices and an overall N(cyt)-C(lum) orientation.

A Salmonella typhimurium Effector Protein SifA Is Modified by Host Cell Prenylation and S-Acylation Machinery

SifA is a Salmonella effector protein that is required for maintenance of the vacuolar membrane that surrounds replicating bacteria. It associates with the Salmonella-containing vacuole but how it interacts with the membrane is unknown. Here we show by immunofluorescence, S100 fractionation and Triton X-114 partitioning that the membrane association and targeting properties of SifA are influenced by a motif encoded within the C-terminal six amino acids. This sequence shares homology with both CAAX and Rab geranylgeranyl transferase prenylation motifs. We characterized the post-translational processing of SifA and showed that the cysteine residue within the CAAX motif is modified by isoprenoid addition through the action of protein geranylgeranyl transferase I. SifA was additionally modified by S-acylation of an adjacent cysteine residue. Similar modifications to host cell proteins regulate numerous functions including protein targeting, membrane association, protein-protein interaction, and signal transduction. This is the only known example of a bacterial effector protein that is modified both by mammalian cell S-acylation and prenylation machinery.

Retinoic acid activates human secretin gene expression by Sp proteins and Nuclear Factor I in neuronal SH‐SY5Y cells

Secretin is a neuropeptide that is expressed in distinct central neurones. As there is no information on how the secretin gene is regulated in neuronal cells, a well established neuronal differentiation cell model, SH-SY5Y, was used to study transcriptional regulation of the human secretin gene. High secretin transcript and peptide levels were found in this cell, and secretin gene expression and promoter activity were up-regulated upon all-trans retinoic acid (RA) treatment. Within the promoter, a functional GC-box 1 (-131 from ATG, relative to the ATG initiation codon) was found to be regulated by a brain-specific Sp protein, Sp4, and ubiquitous factors Sp1 and Sp3. The human secretin gene in SH-SY5Y cells is controlled by the (Sp1 + Sp4)/Sp3 ratio and the RA-induced activation is a partial result of a decrease in Sp3 levels. In addition to the GC-box 1, an N1 motif in close proximity was also responsible for RA-induced secretin gene activation. Competitive gel mobility shift and southwestern blot studies revealed binding of Nuclear Factor I (NFI) with the N1 motif. Overexpression of NFI-C increased promoter activity upon RA treatment. Consistent with this observation, NFI-C transcript levels were augmented after RA treatment. We conclude that RA induction of the secretin gene in neuronal cells is regulated by the combined actions of reducing Sp3 and increasing NFI-C expression.

Identification of alternative promoter usage for the matrix Gla protein gene

Recent cloning of the Xenopus laevis (Xl) matrix Gla protein (MGP) gene indicated the presence of a conserved overall structure for this gene between mammals and amphibians but identified an additional 5'-exon, not detected in mammals, flanked by a functional, calcium-sensitive promoter, 3042 bp distant from the ATG initiation codon. DNA sequence analysis identified a second TATA-like DNA motif located at the 3' end of intron 1 and adjacent to the ATG-containing second exon. This putative proximal promoter was found to direct transcription of the luciferase reporter gene in the X. laevis A6 cell line, a result confirmed by subsequent deletion mutant analysis. RT-PCR analysis of XlMGP gene expression during early development identified a different temporal expression of the two transcripts, strongly suggesting differential promoter activation under the control of either maternally inherited or developmentally induced regulatory factors. Our results provide further evidence of the usefulness of nonmammalian model systems to elucidate the complex regulation of MGP gene transcription and raise the possibility that a similar mechanism of regulation may also exist in mammals.

Elf5 is essential for early embryogenesis and mammary gland development during pregnancy and lactation

Elf5 is an epithelial-specific ETS factor. Embryos with a null mutation in the Elf5 gene died before embryonic day 7.5, indicating that Elf5 is essential during mouse embryogenesis. Elf5 is also required for proliferation and differentiation of mouse mammary alveolar epithelial cells during pregnancy and lactation. The loss of one functional allele led to complete developmental arrest of the mammary gland in pregnant Elf5 heterozygous mice. A quantitative mRNA expression study and Western blot analysis revealed that decreased expression of Elf5 correlated with the downregulation of milk proteins in Elf5(+/-) mammary glands. Mammary gland transplants into Rag(-/-) mice demonstrated that Elf5(+/-) mammary alveolar buds failed to develop in an Elf5(+/+) mammary fat pad during pregnancy, demonstrating an epithelial cell autonomous defect. Elf5 expression was reduced in Prolactin receptor (Prlr) heterozygous mammary glands, which phenocopy Elf5(+/-) glands, suggesting that Elf5 and Prlr are in the same pathway. Our data demonstrate that Elf5 is essential for developmental processes in the embryo and in the mammary gland during pregnancy.

Identification and characterization of novel variants of the thioredoxin reductase 3 new transcript 1 TXNRD3NT1

We have identified and characterized a new gene sequence, TXNRD3NT1, whose transcripts, corresponding to the EST AA430236, were found by Affymetrix DNA chip analysis to be significantly down regulated in affected psoriatic tissue. The full-length cDNA of TXNRD3NT1 was isolated and characterized by combining 5'- and 3'-RACE (rapid amplication of cDNA ends) with screening a keratinocyte cDNA library, designing appropriate PCR primers, cloning amplified products, sequencing, and sequence analysis. Because part of this gene overlaps the previously described thioredoxin reductase 3 (TXNRD3) gene, we have named it TXNRD3NT1 (TXNRD3 new transcript 1). The full-length TXNRD3NT1 cDNA has 1133 nucleotides with a 251-bp 3-UTRand 2 poly(A)signal variants and 2 poly (A) sites. The TXNRD3NT1 cDNA ORF encodes for 133 amino acids, with the first four residues coding for a tubulin-beta mRNA autoregulation signal. Mapping the cDNA nucleotide sequence to the human genome sequence revealed that the TXNRD3NT1 gene has 4 exons located on Chromosome 3, at position 3q21. Exons 1 and 2 of the TXNRD3NT1 gene overlap with exons 15 and 16 of the thioredoxin reductase 2 gene which has different ORFs to that of TXNRD3NT1. The translation initiation codon ATG was found in exon 3 of the TXNRD3NT1 gene. RT-PCR showed that the full-length variant of the TXNRD3NT1 gene was expressed in only four issues (pancreas, esophagus, bone marrow, and keratinocytes) of the 30 different tissues tested. In most other tissues, an aberrant and truncated form of the transcript (i.e., missing exon 3 and part of exon 4) was detected. The result of a preliminary association study between psoriasis and single microsatellite marker of the TXNRD3NT1 gene suggests that it may not be a significant genetic determinant of psoriasis. However, we cannot exclude the possibility that other sequence variants may still exist within the TXNRD3NT1 gene. Sequence analysis of the TXNRD3NT1 gene from 8 psoriasis patients and 8 healthy controls revealed a number of synonymous SNPs that may be useful markers for future disease association studies.

An alternative use of basic pGEX vectors for producing both N- and C-terminal fusion proteins for production and affinity purification of antibodies

Glutathione S-transferase (GST) fusion proteins are widely used in protein production for pure immunogens, protein–protein, and DNA–protein interaction studies. Using basic pGEX vectors, foreign DNA is introduced to the C-terminus of the GST gene and the produced fusion proteins are C-terminally orientated. However, because the orientation of foreign polypeptides may have a very important role in the correct folding of the produced polypeptides, N-terminal fusion proteins are needed to express especially the N-terminus of the foreign polypeptide. Here, we introduce a novel use of the basic pGEX vectors for the production of N-terminal fusion proteins. In this procedure, PCR generated DNA fragments were cloned into the N-terminus of the GST gene in a unique EcoNI site located down-stream of the ATG initiation codon. The N-terminal fusion proteins were expressed in high quantities, easily solubilized, and affinity purified using our modification of current purification protocols. We also introduce here a new modification of the affinity purification of antibodies using covalently crosslinked GST and fusion proteins to glutathione–agarose beads. Our procedure was tested successfully for producing antibodies against both N- and C-terminus of the luteinizing hormone/chorionic gonadotropin receptor.

Variable phenotype related to a novel PAX 6 mutation (IVS4+5G>C) in a family presenting congenital nystagmus and foveal hypoplasia

Several ocular defects have been identified as a consequence of the PAX6 gene mutations. With regard to the implication of this gene in unusual phenotypes, we report a family presenting with congenital nystagmus, foveal hypoplasia, and iris hypoplasia or atypical coloboma. Observational case report. The entire transcribed region of the PAX6 gene was submitted to mutation search at the DNA and mRNA levels in five affected members of a French family in test with 82 normal subjects. A novel heterozygous PAX6 gene splice mutation (IVS4 + 5G>C) was identified. The mutation is located in IVS4 within the consensus donor splice site. A mutant mRNA lacking exon 4 as the sole defect was evidenced. The resultant protein was predicted to contain a cryptic ATG initiation codon in exon 3 and a slightly altered paired-domain in an open reading frame extended by 13 amino acids. The association of anterior segment anomalies and foveal hypoplasia with one of the slightest alterations of the PAX6 protein described to date confirms the association of variant phenotypes with hypomorphic alleles. Mutation screening of the PAX6 gene could be useful in elucidating the origin of complex ocular malformations.