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PubMed1997

A high-resolution PAC and BAC map of the SCA2 region.

The spinocerebellar ataxia type 2 (SCA2) gene has been localized to chromosome 12q24.1. To characterize this region and to aid in the identification of the SCA2 gene, we have constructed a 3.9-Mb physical map, which covers markers D12S1328 and D12S1329 known to flank the gene. The map comprises a contig of 84 overlapping yeast artificial chromosomes (YACs), P1 artificial chromosomes (PACs), and bacterial artificial chromosomes (BACs) onto which we placed 82 PCR markers. We localized eight genes and expressed sequence tags on this map, many of which had not been precisely mapped before. In contrast to YACs, which showed a high degree of chimerism and deletions in this region, PACs and BACs were stable. Only 1 in 65 PACs contained a small deletion, and 2 in 18 BACs were chimeric. The high-resolution physical map, which was used in the identification of the SCA2 gene, will be useful for the positional cloning of other disease genes mapped to this region.

Ataxins↗
PubMed2000

Gene structures and expression profiles of three human KCND (Kv4) potassium channels mediating A-type currents I(TO) and I(SA).

The four known members of the KCND/Kv4 channel family encode voltage-gated potassium channels. Recent studies provide evidence that members of the Kv4 channel family are responsible for native, rapidly inactivating (A-type) currents described in heart (I(TO)) and neurons (I(SA)). In this study, we cloned the human KCND1 cDNA, localized the KCND1 gene to chromosome Xp11.23-p11.3, and determined the genomic structure and tissue-specific expression of the KCND1, KCND2, and KCND3 genes, respectively. The open reading frame of Kv4. 1 is 1941 nucleotides long, predicting a protein of 647 amino acids. The deduced protein sequence of Kv4.1 shows an overall identity of 60% with Kv4.2 and Kv4.3L and corresponds to the common structure of voltage-gated potassium channels. KCND1-specific transcripts were detectable in human brain, heart, liver, kidney, thyroid gland, and pancreas, as revealed by Northern blot and RT-PCR experiments. The comparison of the expression patterns of the known Kv4 family members shows subtype specificity with significant overlaps. The KCND gene structures exhibit an evolutionarily conserved exon pattern with a large first exon containing the intracellular N-terminus and the putative membrane-spanning regions S1 to S5, as well as part of the pore region. The KCND3 gene contains an additional exon of 57 bp, which is not present in the other two KCND genes and gives rise to the C-terminal splice KCND3L variant with an insertion of 19 amino acids.

Chromosome Mapping↗
PubMed2001

Axonemal beta heavy chain dynein DNAH9: cDNA sequence, genomic structure, and investigation of its role in primary ciliary dyskinesia.

Dyneins are multisubunit protein complexes that couple ATPase activity with conformational changes. They are involved in the cytoplasmatic movement of organelles (cytoplasmic dyneins) and the bending of cilia and flagella (axonemal dyneins). Here we present the first complete cDNA and genomic sequences of a human axonemal dynein beta heavy chain gene, DNAH9, which maps to 17p12. The 14-kb-long cDNA is divided into 69 exons spread over 390 kb. The cDNA sequence of DNAH9 was determined using a combination of methods including 5' rapid amplification of cDNA ends, RT-PCR, and cDNA library screening. RT-PCR using nasal epithelium and testis RNA revealed several alternatively spliced transcripts. The genomic structure was determined using three overlapping BACs sequenced by the Whitehead Institute/MIT Center for Genome Research. The predicted protein, of 4486 amino acids, is highly homologous to sea urchin axonemal beta heavy chain dyneins (67% identity). It consists of an N-terminal stem and a globular C-terminus containing the four P-loops that constitute the motor domain. Lack of proper ciliary and flagellar movement characterizes primary ciliary dyskinesia (PCD), a genetically heterogeneous autosomal recessive disorder with respiratory tract infections, bronchiectasis, male subfertility, and, in 50% of cases, situs inversus (Kartagener syndrome, KS). Dyneins are excellent candidate genes for PCD and KS because in over 50% of cases the ultrastructural defects of cilia are related to the dynein complex. Genotype analysis was performed in 31 PCD families with two or more affected siblings using a highly informative dinucleotide polymorphism located in intron 26 of DNAH9. Two families with concordant inheritance of DNAH9 alleles in affected individuals were observed. A mutation search was performed in these two "candidate families," but only polymorphic variants were found. In the absence of pathogenic mutations, the DNAH9 gene has been excluded as being responsible for autosomal recessive PCD in these families.

Adenosine Triphosphate↗
PubMed2005

Determination of antihypertensive mixtures by use of a chemometrics-assisted spectrophotometric method.

A simple multivariate calibration method for analysis of two types of hypotensive mixture is described. The mixtures are composed of chlorthalidone with atenolol or chlorthalidone with both amiloride hydrochloride and atenolol. The components of the mixtures result in substantial spectral overlap-between 87.5 and 91.0%. Resolution of the mixtures under investigation has been accomplished mainly by using CLS and PCR methods. The components in each mixture have been simultaneously determined in three commercial dosage forms with high accuracy and without interference from commonly encountered excipients and additives. Good recoveries were obtained with both synthetic mixtures and commercial tablets. The results obtained were compared with those from pharmacopeial methods and found to be in good agreement. The results obtained from CLS and PCR were also compared with those obtained from a 1D spectrophotometric method.

Amiloride↗
PubMed1994

Isolation and sequence of an endochitinase-encoding gene from a cDNA library of Trichoderma harzianum.

There are no reports of gene sequences coding for extracellular chitinolytic enzymes from filamentous fungi, even though these enzymes are considered critical to the biological control of plant pathogenic fungi. The purpose of this paper was to report the isolation of a gene (ThEn-42) encoding endochitinase (Ech) from Trichoderma harzianum strain P1, describe its sequence, and to determine whether it was related to genes coding for enzymes with similar functions from prokaryotic or other eukaryotic sources. A clone containing a 1096-bp foreign cDNA fragment was isolated from thalli grown under induced conditions. This cDNA molecule was sequenced and found to lack a portion of the 5' terminus. Polymerase chain reaction (PCR) was used to isolate a fragment from the lambda gt11 library which contained the 5' terminus plus an overlap region with the 1096-bp cDNA clone. The full-length cDNA sequence, consisting of 1554 bp, contained an open reading frame (ORF) expressing a protein of 424 amino acids (aa). Southern analysis of genomic DNA indicated that there is only a single gene in strain P1 with sequence identity to the sequence described in this report. One region within the protein, thought to be required for catalytic activity of the enzyme, was highly conserved between genes coding for Ech from Th, Serratia marcescens, Bacillus circulans, Streptomyces plicatus, Vibrio parahemolyticus and Kluyveromyces lactis.

Amino Acid Sequence↗
PubMed2006

The last Viking King: a royal maternity case solved by ancient DNA analysis.

The last of the Danish Viking Kings, Sven Estridsen, died in a.d. 1074 and is entombed in Roskilde Cathedral with other Danish kings and queens. Sven's mother, Estrid, is entombed in a pillar across the chancel. However, while there is no reasonable doubt about the identity of Sven, there have been doubts among historians whether the woman entombed was indeed Estrid. To shed light on this problem, we have extracted and analysed mitochondrial DNA (mtDNA) from pulp of teeth from each of the two royals. Four overlapping DNA-fragments covering about 400bp of hypervariable region 1 (HVR-1) of the D-loop were PCR amplified, cloned and a number of clones with each segment were sequenced. Also a segment containing the H/non-H specific nucleotide 7028 was sequenced. Consensus sequences were determined and D-loop results were replicated in an independent laboratory. This allowed the assignment of King Sven Estridsen to haplogroup H; Estrid's sequence differed from that of Sven at two positions in HVR-1, 16093T-->C and 16304T-->C, indicating that she belongs to subgroup H5a. Given the maternal inheritance of mtDNA, offspring will have the same mtDNA sequence as their mother with the exception of rare cases where the sequence has been altered by a germ line mutation. Therefore, the observation of two sequence differences makes it highly unlikely that the entombed woman was the mother of Sven. In addition, physical examination of the skeleton and the teeth strongly indicated that this woman was much younger (approximately 35 years) at the time of death than the 70 years history records tell. Although the entombed woman cannot be the Estrid, she may well be one of Sven's two daughters-in-law who were also called Estrid and who both became queens.

Base Sequence↗
PubMed2006

Current diagnostic criteria for the chronic myeloproliferative disorders (MPD) essential thrombocythemia (ET), polycythemia vera (PV) and chronic idiopathic myelofibrosis (CIMF).

The clinical criteria for the diagnosis of essential thrombocythemia (ET) according to the polycythemia vera study group (PVSG) do not distinguish between ET and thrombocythemia associated with early stage PV and prefibrotic chronic idiopathic myelofibrosis (CIMF). The clinical criteria of the PVSG for the diagnosis of polycythemia vera (PV) only detects advanced stage of PV with increased red cell mass. The bone marrow criteria of the World Health Organization (WHO) are defined by pathologists to explicitly define the pathological criteria for the diagnostic differentiation of ET, PV, and prefibrotic and fibrotic CIMF. As the clinical PVSG and the pathological WHO criteria show significant shortcomings, an updated set of European Clinical and Pathological (ECP) criteria combined with currently available biological and molecular markers are proposed to much better distinct true ET from early PV mimicking ET, to distinguish ET from thrombocythemia associated with prefibrotic CIMF, and to define the various clinical and pathological stages of PV and CIMF that has important therapeutic and prognostic implications. Comparing the finding of clustered giant abnormal megakaryocytes in a representative bone marrow as a diagnostic clue to MPD, the sensitivity for the diagnosis of MPD associated with splanchnic vein thrombosis was 63% for increased red cell mass, 52% for low serum EPO level, 72% for EEC, and 74% for splenomegaly indicating the superiority of bone marrow histopathology to detect masked early and overt MPD in this setting. The majority of PV and about half of the ET patients have spontaneous EEC, low serum EPO levels and PRV-1 over-expression and are JAK2 V617F positive. The positive predictive value for the diagnosis of PV of spontaneous growth of endogenous erythroid colonies (EEC) of peripheral blood (PB) and bone marrow (BM) cells is about 80-85% when either PB or BM EEC assays, and up to 94% when BM and PB EEC assays were performed. The diagnostic impact of low serum EPO levels (ELISA assay) in a large study of 186 patients below the normal range (<3.3 IU/l) had a sensitivity specificity and positive predictive value of 87%, 97% and 97.8%, respectively, for the diagnosis of PV. There is a significant overlap of serum EPO levels in PV versus control and controls versus SE. The specificity of a JAK2 V617F PCR test for the diagnosis of MPD is high (near 100%), but only half of ET and MF (50%) and the majority of PV (up to 97%) are JAK2 V617F positive. The use of biological markers including JAK2 V617 PCR test, serum EPO, PRV-1, EEC, leukocyte alkaline phosphatase score and peripheral blood parameters combined with bone marrow histopathology has a high sensitivity and specificity (almost 100%) to diagnose the early and overt stages of ET, PV and CIMF in JAK2 V617F positive and negative MPDs.

Amino Acid Substitution↗
PubMed2004

Molecular cloning of proopiomelanocortin cDNA in the ratfish, a holocephalan.

Proopiomelanocortin (POMC) is a precursor for several pituitary hormones including adrenocorticotropin (ACTH), melanocyte-stimulating hormone (MSH) and endorphin (END). Fish POMCs in four taxonomic classes, Cephalaspidomorphi (lampreys), Chondrichthyes (cartilaginous fish), Sarcopterygii (lobe-finned fish), and Actinopterygii (ray-finned fish) have been identified. However, two essential species, ratfish in Chondrichthyes and hagfish in Agnatha, are still missing in the evolutionary image of this molecule. The present study reports analysis of POMC cDNA in the ratfish, Chimaera phantasma, which belongs to another subclass in the Chondrichthyes. Partial cDNA clones were amplified by PCR from single-strand cDNA prepared on total RNA from a complex of pituitary and hypothalamus, and subsequently overlapped to obtain a full-length sequence. Ratfish POMC cDNA consists of 1294bp excluding the poly(A) tail. It encodes a signal peptide of 25 amino acids and POMC of 300 amino acids. gamma-MSH, ACTH, alpha-MSH, delta-MSH, beta-MSH, and beta-END are located at prePOMC (76-87), (120-158), (120-132), (212-227), (275-290), and (293-325), respectively. delta-MSH, originally found in elasmobranch POMCs, was also present in ratfish POMC, suggesting this structure might have appeared after the divergence of chondrichthians from the ancestral lineage. Thus, we demonstrated the common occurrence of four MSHs in chondrichthian POMC and established a clear understanding of the molecular evolution of POMC in gnathostomes.

Amino Acid Sequence↗
PubMed2003

The complete mitochondrial genome of the Chinese giant salamander, Andrias davidianus (Amphibia: Caudata).

The mitochondrial genome of the Chinese giant salamander Andrias davidianus was isolated using the long-and-accurate polymerase chain reaction (LA PCR) method. The sequencing work adopted the shotgun strategy accompanying with seven internal primers to cover the gaps where overlapping clones were not available. The entire mtDNA sequence is 16,503 bp long, with a gene content of 13 protein-coding, two ribosomal RNA and 22 transfer RNA genes, and order identical to that observed in most other vertebrates except for an additional 318 bp non-coding sequence between tRNA-Thr and tRNA-Pro genes. In order to carry out molecular phylogenetic analyses, all 13 protein sequences deduced from whole mitochondrial genomes for eight vertebrate species (six amphibians, two lobe-finned fishes) were combined to a single data set. This data set was refined by the program Gblocks using a stringent parameter setting and then subjected to MP, ML and NJ analyses. Thus phylogenetic relationships among living amphibians were discussed.

Animals↗
PubMed2001

Identification and molecular characterisation of the complete genome of a Singapore isolate of hepatitis C virus: sequence comparison with other strains and phylogenetic analysis.

The complete genomic sequence of a Singapore isolate of the hepatitis C virus (HCV) was obtained from serum of an individual chronically infected with HCV. Nine overlapping cDNA clones covering the entire viral genome was amplified by reverse-transcription-polymerase chain reaction (RT-PCR), This isolate (HCV-S1) comprised 9,609 nucleotides (nt), including 341 nt of the complete 5' untranslated region (5' UTR), a single open reading frame of 3,011 amino acids (aa) and 235 nt of the complete 3' UTR. Its genotype was identified as type lb from analyses of its sequences in the 5' UTR, NS3 and NS5B regions. When compared against nine reported HCV isolates, the overall aa homology of HCV-SI was closest with an Australian strain, HCV-A (94%) and a Japanese strain, HCV-JT (93.9%). Phylogenetic analysis revealed that it was most closely related to the Taiwan strain, HCV-TW and another Japanese strain, HCV-K1-R1.

5' Untranslated Regions↗
PubMed2024

Mosquitoes provide a transmission route between possums and humans for Buruli ulcer in southeastern Australia.

Buruli ulcer, a chronic subcutaneous infection caused by Mycobacterium ulcerans, is increasing in prevalence in southeastern Australia. Possums are a local wildlife reservoir for M. ulcerans and, although mosquitoes have been implicated in transmission, it remains unclear how humans acquire infection. We conducted extensive field survey analyses of M. ulcerans prevalence among mosquitoes in the Mornington Peninsula region of southeastern Australia. PCR screening of trapped mosquitoes revealed a significant association between M. ulcerans and Aedes notoscriptus. Spatial scanning statistics revealed overlap between clusters of M. ulcerans-positive Ae. notoscriptus, M. ulcerans-positive possum excreta and Buruli ulcer cases, and metabarcoding analyses showed individual mosquitoes had fed on humans and possums. Bacterial genomic analysis confirmed shared single-nucleotide-polymorphism profiles for M. ulcerans detected in mosquitoes, possum excreta and humans. These findings indicate Ae. notoscriptus probably transmit M. ulcerans in southeastern Australia and highlight mosquito control as a Buruli ulcer prevention measure.

Animals↗
PubMed1997

Reduced expression and normal nucleotide sequence of androgen receptor gene coding and promoter regions in a family with partial androgen insensitivity syndrome.

BACKGROUND AND OBJECTIVES: Androgen insensitivity syndrome (AIS) is an X-linked disorder of XY males characterized by varying degrees of impaired masculinization. In many AIS cases, mutations have been identified in the coding sequence of the human androgen receptor (AR) gene which impair receptor function. Cases have also been reported in which reduced AR mRNA expression may contribute to AIS in association with AR gene mutations. The purpose of this study was to define the molecular basis of AIS in members of a family with clinical and laboratory features of partial androgen insensitivity (PAIS). DESIGN: Genital skin fibroblast (GSF) cultures were established from foreskin tissue for androgen receptor binding analysis. Genomic DNA was obtained from blood leucocytes for AR gene nucleotide sequence analysis. AR mRNA levels were determined in total RNA extracted from GSF cultures. PATIENTS: Three related subjects with perineo-scrotal hypospadias, bifid scrotum and microphallus were studied. The family pedigree of these subjects suggested an X-linked pattern of inheritance. Hormone assay results were consistent with AIS. MEASUREMENTS: AR binding capacity and affinity were determined in three subjects and compared with unaffected male controls. The coding sequence and 1.4 kb of promoter region of the AR gene were amplified in overlapping fragments by polymerase chain reaction from genomic DNA and sequenced. GSF AR mRNA was measured by a competitive PCR technique. RESULTS: In the PAIS subjects, AR affinity in cultured GSF was normal (Kd = 0.24, 0.30, 0.48 vs 0.27 +/- 0.07 (SD) nmol/l) but binding capacity was reduced (Bmax = 0.31, 0.36, 0.27 vs 1.26 +/- 0.37 (SD) fmol/microgram DNA). Sequence analysis of the CAG repeat polymorphism within exon 1 of the AR gene showed that both mothers were heterozygous at this locus, and that the three subjects had inherited the same allele. GSF AR mRNA levels were reduced in all three patients compared with controls (0.25, 0.74 and 0.74 vs 3.8 +/- 0.9 (SEM), range 1.8-7.3 amol/microgram total RNA). The nucleotide sequences of the entire AR coding region and of a 1.4 kb segment containing the promoter region were normal. CONCLUSION: Members of this family with clinical and biochemical evidence of X-linked partial androgen insensitivity syndrome demonstrated normal androgen receptor binding affinity and androgen receptor gene nucleotide sequence but reduced androgen receptor binding capacity and reduced androgen receptor mRNA. These results suggest that partial androgen insensitivity syndrome in this family may be caused by reduced expression of a normal androgen receptor gene.

Cells, Cultured↗
PubMed1998

Determination of allele frequency from DNA pools using bovine trinucleotide microsatellites.

Three sets of fluorescent labelled primers were used to amplify bovine trinucleotide microsatellites from DNA pools. DNA from 20 individuals were collected to create 3 pools differing in allele frequencies. Replicate mixes from each pool were used as template for PCR reactions. PCR products were separated and quantified on an automated DNA sequencer. Allele frequency estimates from pooled samples corrected for overlapping shadow peaks were calculated. Rare alleles representing only 2.5% of the total pool were accurately detected. Standard error of allele frequency estimates expressed as percent of the total 40 chromosomes per pool ranged between 0.8%-4.6% for different microsatellite-pool combinations as compared to 8.0% binomial sampling error. Regression coefficients of actual allele frequencies, determined by individual genotyping, on estimated frequencies ranged from 0.96-1.06. As regression slopes were close to unity it can be deduced that corrected peak height values from a DNA pool are unbiased estimates of actual allele frequencies. With standard error of the y-intercept of 0.21, the 95% confidence interval of allele frequency is 0.42 alleles or 1% in a pool of 40 chromosomes. Thus, it would be possible to detect an allele with a frequency of greater than 1% within the pool.

Alleles↗
PubMed1998

Pseudomonas aeruginosa PAO1 bacterial artificial chromosomes: strategies for mapping, screening, and sequencing 100 kb loci of the 5.9 Mb genome.

Pseudomonas aeruginosa is an opportunistic bacterial pathogen frequently found in nosocomial infections and is a major cause of morbidity and mortality in patients with cystic fibrosis. To facilitate molecular studies of this organism, we have generated a bacterial artificial chromosome (BAC) library. Genomic DNA was isolated from the prototype strain PAO1, partially digested with HindIII, size selected after pulsed-field gel electrophoresis, and used to construct a BAC library using the pBeloBAC11 vector. DNAs from approximately 850 clones, representing more than 9.5-fold physical coverage of the 5.9-Mb PAO1 genome, were analyzed after SpeI and HindIII digestions and agarose gel electrophoresis. The BAC library had clones with insert fragments ranging from 20 to more than 290 kb. A subset of 264 BACs having inserts > 80 kb, representing > 4 genome equivalents, were rearrayed into 96-well plates, and a clone pooling and PCR screening strategy was developed. The PCR library screening enabled the identification and recovery of BACs containing genes implicated in cell division and in cell wall biosynthesis, as well as a series of known genes mapping to different regions of the PAO1 chromosome. A physical and genetic map was constructed for the 98-kb pMOC5 BAC clone, which spans the entire fts-mur locus. Chromosome walking from each end of the pMOC5 clone placed it within a contig spanning 243 kb. The BAC library and screening resources now allow a PCR-based screening of a P. aeruginosa genomic library for any gene of interest. The restriction fragment analysis of overlapping clones indicated that BAC clones stably maintain and propagate Pseudomonas DNA, providing evidence that the PAO1 BAC library is an appropriate reagent for genome sequencing.

Bacterial Proteins↗
PubMed1996

Proliferative T cell responses to human papillomavirus type 16 L1 peptides in patients with cervical dysplasia.

Human papillomavirus type 16 (HPV-16) can cause genital warts, cervical dysplasias and carcinoma of the cervix. Cell-mediated immunity is thought to be important in protection against the virus and in its elimination, but little is known about the mechanisms involved. In a cross-sectional study we have demonstrated proliferative T cell responses to peptides representing the HPV-16 L1 capsid protein (aa 199-409) in the peripheral blood of 63% of patients (n = 41) with histological evidence of cervical dysplasia and in 45% of healthy age-matched controls (n = 11). This was achieved by generating short-term T cell lines (STLs) from each individual in vitro against a beta-galactosidase-HPV- 16 L1 (aa 199-409) fusion protein for 2 weeks, and then identifying the HPV epitopes they recognized with overlapping synthetic peptides (15-mers) spanning this region in 3 day specificity assays. Histological grading and HPV typing by PCR were performed on patients' cervical biopsies taken at the same clinical visit as the peripheral blood samples. An immunogenic region was identified between aa 311-345 in 73% of patients (18% in controls) who responded to HPV-16 L1 (aa 199-409). The number of responders to this region was significantly higher in patients with HPV-16-positive biopsies when compared to those with HPV-16-negative biopsies (P = 0.006), as was the number of responders to individual peptides 311-325 (NLASSNYFPTPSGSM; p = 0.04) and 321-335 (PSGSMVTSDAQIFNK; P = 0.004) representing this region. The mean level of response to each individual peptide was also higher in the patient group than the controls (P < 0.05). The most significant finding was that all patients with evidence of a current HPV-16 infection responded to one or more L1 peptides (P = 0.0004) and 92% had high grade cervical intraepithelial neoplasia (CIN III). We also found that the CIN III group was more likely to respond to any L1 peptide than either the atypical group (P = 0.04) or the controls (P = 0.05). Data from four individuals showed that the majority of peptide-specific STLs were CD4+ but some CD8+ STLs were also detected.

Amino Acid Sequence↗
PubMed2002

Characterization of Spodoptera exigua multicapsid nucleopolyhedrovirus ORF17/18, a homologue of Xestia c-nigrum granulovirus ORF129.

Spodoptera exigua multicapsid nucleopolyhedrovirus (SeMNPV) contains a number of genes with a homologue found so far only in a distantly related baculovirus. One of these, SeMNPV ORF17/18 (Se17/18) shares 55% amino acid similarity to ORF129 of Xestia c-nigrum granulovirus (XcGV). Se17/18 was transcribed in cultured S. exigua 301 cells, as a polyadenylated transcript of 1.1 kb. 5'-RACE analysis demonstrated that Se17/18 transcripts started at 134, 131 and 126 nt upstream of the putative translational start codon. These sites overlap with a baculovirus consensus early promoter motif. Se17/18 transcripts were detected by Northern blot analysis and RT-PCR with increasing abundance from 8 h to 24 h post infection (p.i.) and still present until 72 h p.i. A C-terminal GFP-fusion protein of Se17/18 was primarily localized in the cytoplasm of Se301 and Sf21 cells. A chicken polyclonal antiserum was raised that reacted specifically to Se17/18 protein produced in E. coli. However, no immunoreactive protein was detected in SeMNPV-infected Se301 cells and S. exigua larvae, neither in concentrated BV and ODV preparations. These observations and the inability to detect a C-terminal GFP-fusion protein of Se17/18 in Se301 cells using a GFP antibody suggest that Se17/18 protein is present, if at all, in spurious amounts. Based on the low homology of the Se17/18 protein to (methyl) transferases its possible involvement in transcription regulation is discussed.

Amino Acid Sequence↗
PubMed1998

Cloning and functional expression of rat ether-à-go-go-like K+ channel genes.

1. Screening of rat cortex cDNA resulted in cloning of two complete and one partial orthologue of the Drosophila ether-à-go-go-like K+ channel (elk). 2. Northern blot and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis revealed predominant expression of rat elk mRNAs in brain. Each rat elk mRNA showed a distinct, but overlapping expression pattern in different rat brain areas. 3. Transient transfection of Chinese hamster ovary (CHO) cells with rat elk1 or rat elk2 cDNA gave rise to voltage-activated K+ channels with novel properties. 4. RELK1 channels mediated slowly activating sustained potassium currents. The threshold for activation was at -90 mV. Currents were insensitive to tetraethylammonium (TEA) and 4-aminopyridine (4-AP), but were blocked by micromolar concentrations of Ba2+. RELK1 activation kinetics were not dependent on prepulse potential like REAG-mediated currents. 5. RELK2 channels produced currents with a fast inactivation component and HERG-like tail currents. RELK2 currents were not sensitive to the HERG channel blocker E4031.

Amino Acid Sequence↗
PubMed2004

Identification of new targets of Drosophila pre-mRNA adenosine deaminase.

Adenosine deaminase acting on RNA (ADAR) in Drosophila and mammals has recently become the target of numerous investigations. It is now clear that this protein has a number of functions in the nervous system. Indeed, the mutation of ADAR in Drosophila (dADAR) results in many pathological and physiological changes, such as sensitivity to hypoxia and neuronal degeneration. To understand the full scope of dADAR function, it is crucial to identify new dADAR targets. A polyclonal antibody against inosine was developed and used to enrich inosine-containing mRNAs. The efficiency of immunoaffinity purification was confirmed for the Q/R editing site of GluR-B pre-mRNA that has been edited by ADAR2 to generate inosines at the editing site. This approach was applied to enrich inosine-containing mRNAs from total mRNAs of wild-type and dADAR mutant flies, respectively. The enriched mRNA portion was then amplified and hybridized with Drosophila cDNA arrays. With this method, over 500 mRNAs were identified as potential dADAR targets by showing a higher amount in the enriched mRNA portion from wild-type flies than from dADAR mutant flies. The occurrence of A-to-G conversion in these mRNAs was further analyzed by comparing over 7,000 Drosophila cDNAs sequences with their genomic sequences. A final list of 62 candidates was generated from the overlap of the two approaches. Twelve genes from the final list were further examined by sequencing the RT-PCR products of these genes from wild-type and dADAR mutant flies. Seven of the 12 genes were proven to have A-to-G changes in the wild-type but not in mutant flies. We conclude that the combination of immunoaffinity enrichment of inosine-containing mRNA, DNA microarrays, and sequence comparison could facilitate the discovery of new dADAR substrates, which in turn allows us to better understand the targets of dADAR and the biological function of A-to-I RNA editing in flies.

Adenosine Deaminase↗