Isolation and characterization of cDNA clones encoding a 32-kDa dense-granule antigen of Sarcocystis muris (Apicomplexa)

A monoclonal antibody (2F4) directed against a 32-kDa dense-granule antigen of Sarcocystis muris cyst merozoites (bradyzoites) was used to screen a lambda ZAP cDNA expression library. A clone with an insert of 1.4 kb in length (DG 32/1) was isolated. A fusion protein derived from bacteria harbouring the recombinant plasmid DG 32/1 reacted with monoclonal antibody (mAb) 2F4. Southern blot hybridization suggests that the gene is present as a single copy. On Northern blots, a single mRNA species of 1.8 kb was detected by a cDNA-derived probe. In addition, we isolated a full-length clone (DG 32/PH1) by screening the cDNA library with a non-radioactive-labelled cDNA probe. The nucleotide sequence of DG 32/PH1 comprises 1.57 kb. It contains an open reading frame of 882 bp with a coding capacity of approximately 32 kDa. The hypothetical polypeptide consists of a putative N-terminal signal peptide and the mature protein sequence. The occurrence of an N-terminal signal sequence is consistent with the observation that the 32-kDa protein of S.␣muris is secreted from the dense granules. Received: 12 December 1997 / Accepted: 15 January 1998     

Amplification of genomic DNA fragments of Sarcocystis muris (Apicomplexa) cyst merozoites encoding a thiol (cysteine) proteinase

Parasites of the phylum Apicomplexa (Sporozoa) cause diseases such as malaria, toxoplasmosis, or intestinal coccidiosis. Invasive stages possess typical apical organelles such as dense granules that harbor a broad range of polypeptides that are believed to take part in the parasite-host cell interaction. In previous studies a 26-kDa polypeptide of dense granules from Sarcocystis muris cyst merozoites (bradyzoites) was characterized as a thiol (cysteine) proteinase. In this paper a method is demonstrated to amplify DNA fragments from genomic DNA of S. muris cyst merozoites by polymerase chain reaction, which probably code for the 26-kDa antigen. Received: 12 December 1997 / Accepted: 17 January 1998     

A novel cDNA clone encoding a specific excretory/secretory antigen of larval Trichinella pseudospiralis

A cDNA library for Trichinella pseudospiralis was constructed to study the expression of specific antigens. Four positive clones were identified using antibodies against the excretory/secretory (ES) products of the nematode as probe. Sequence analysis showed that they contained identical cDNA inserts of 606 bp, including a 5′ non-translated region of 96 bp, a core translated segment of 408 bp and a poly(A)⁺ 3′ terminus. It encoded a novel 136-amino-acid polypeptide. Southern blot analysis indicated that the cDNA did not cross-hybridize to the genomic digests of T. spiralis, mouse, or rat. A single copy only of its complementary sequence was found in the genome of T. pseudospiralis. Using the lambda ZAP expression system, the cDNA was induced to express a 23-kDa β-galactosidase-fusion protein which did not cross-react with polyclonal and monoclonal antibodies against T. spiralis, heat shock proteins, or four heterologous species of nematodes. The antiserum against the fusion protein recognized a 15-kDa band from the ES products of T. pseudospiralis in immunoblotting. Immunocytolocalization demonstrated that the anti-fusion protein serum only recognized an epitope in the stichosome of T. pseudospiralis and not in T. spiralis. The protein can therefore serve as a specific antigen for the differential diagnosis of trichinellosis. Received: 22 December 1998 / Accepted: 17 February 1999     

Isolation and characterization of a cDNA clone encoding a thiol proteinase of Sarcocystis muris cyst mero` zoites (Apicomplexa)

A cDNA library of Sarcocystis muris cyst merozoites was screened using a digoxigenin-labeled probe. This probe was derived from a 504-bp polymerase-chain-reaction fragment representing part of a thiol proteinase. Several cDNA clones were isolated, one of which (PH08) consists of a nucleotide sequence of 1694 bp and encodes the complete prepropolypeptide of a cathepsin L-like proteinase. PH08 contains an open reading frame of 394 amino acid (aa) residues with a 46-residue signal sequence, which is followed by a 129-residue propeptide and 219 aa residues of the mature enzyme. Two potential glycosylation sites and a putative polyadenylation signal were also identified. The occurrence of the highly conserved interspersed ERFNIN aa motif, not found in cathepsin B-like proteinases, suggests the classification of the enzyme as a cathepsin L-like proteinase. Results worked out in this study will enable production of the recombinant thiol proteinase of S. muris cyst merozoites necessary for study of the substrate specificity as well as other biochemical parameters of this enzyme. Received: 20 January 1999 / Accepted: 3 February 1999     

Double deletions and missense mutations in the first nucleotide-binding fold of the ATP-binding cassette transporter A1 (<Emphasis Type="Italic">ABCA1</Emphasis>) gene in Japanese patients with Tangier disease

 Tangier disease (TD) is a rare autosomal recessive disease characterized by plasma high-density lipoprotein deficiency caused by an ATP-binding cassette transporter A1 (ABCA1) gene mutation. We describe three different mutations in Japanese patients with TD. The first patient was homozygous for double deletions of 1221 bp between intron 12 and 14 and 19.9 kb between intron 16 and 31. The breakpoint sequence analyses suggest that it is a simultaneous event caused by double-loop formation through multiple Alu. The second patient was homozygous for a novel mutation of A3198C in exon 19, resulting in Asn935His. The third patient was homozygous for A3199G of exon 19 that leads to Asn935Ser, which is the same mutation found in German and Spanish families. Both Asn mutations involved Walker A motif of the first nucleotide-binding fold. Received: January 24, 2002 / Accepted: March 11, 2002     

Molecular characterization of a satellite RNA associated with blackcurrant reversion nepovirus

Summary.  A satellite RNA (satRNA) associated with blackcurrant reversion nepovirus (BRV) was isolated and its nucleotide sequence was determined from cDNA clones. BRV satRNA was 1432 nucleotides (nt) in length excluding the poly(A)-tail, and contained one open reading frame which encodes a polypeptide of 402 amino acids, with a calculated M_r of 44 220. The coding region was bordered by a 5′ leader sequence of 25 nt and a 3′-nontranslated region of 201 nt. Two in vitro translation products of approximately 45 kDa and 40 kDa were detected, indicating that two in-frame AUG codons at positions 26 and 134 may both be functional. Nucleotide sequence comparisons revealed a stretch of 865 nt that was 63% identical between BRV satRNA and the large satRNA of chicory yellow mottle nepovirus. A 5′-terminal consensus sequence and a 40 nt motif (located at positions 264–303 of BRV satRNA) were conserved between BRV satRNA and other nepoviral large satRNAs. Received March 22, 1999/Accepted August 30, 1999     

Cloning and heterologous expression of Solorina crocea pyrG

A pyrG gene, encoding orotidine 5′-monophosphate decarboxylase, was cloned from a phage library derived from the lichen Solorina crocea. Phylogenetic analysis and a survey of geographically well-separated specimens were used to verify that the gene represented the fungal component of the lichen. Both coding and upstream sequences of S. crocea pyrG exhibited features typical of fungal genes. A 132-bp intron interrupting the coding region between nucleotides 157 and 288 was confirmed by RT-PCR and sequencing. Transformation of Aspergillus nidulans with S. crocea pyrG, controlled by either its native promoter or the A. nidulans trpC promoter, resulted in uridine-independent strains that exhibited appreciable growth only at 24 °C. Southern analysis indicated multiple integrations of S. crocea pyrG. These results demonstrate that heterologous expression may be used to investigate genes from lichens. Received: 1 December 1999 / 10 February 2000     

Isolation and localization of an IDDMK1,2-22-related human endogenous retroviral gene, and identification of a CA repeat marker at its locus

We intended to confirm genetically the involvement of the IDDMK1,2-22 gene in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). For this purpose, we isolated a human endogenous retrovirus gene, possibly corresponding to IDDMK1,2-22. The isolated gene showed 99.8% and 99.7% homologies in nucleotide sequences to a part of the env region and of the 3′-LTR region, respectively, compared to those of IDDMK1,2-22 deposited in GenBank. The gene also showed a close relation to HERV-K18, of which the 3′-LTR sequence gave 99.5% homology. It seemed likely that these genes represented the same single gene. The newly isolated gene was present in the first intron of the CD48 gene and was located on chromosome 1q21.2–q22. A CA repeat marker was found approximately 20 kb upstream from the 5′-end of the 5′-LTR of the gene. Received: March 29, 1999 / Accepted: May 21, 1999     

Effect of +36T > C in intron 1 on the glutamine: fructose-6-phosphate amidotransferase 1 gene and its contribution to type 2 diabetes in different populations

Glutamine: fructose-6-phosphate amidotransferase 1 (GFPT1) acts as a rate-limiting enzyme in the hexosamine biosynthetic pathway, which is an alternative branch of glucose metabolism. To evaluate GFPT1 as a susceptibility gene to type 2 diabetes, we surveyed the polymorphisms related with the gene function of GFPT1 and assessed its contribution to type 2 diabetes with a case-control association study. Screening of the 5′-flanking and all coding regions of GFPT1 revealed eight polymorphisms, one in the 5′-flanking region, one synonymous polymorphism in exon 8, five in introns and one in 3′-UTR, but no mis-sense or non-sense polymorphism. With in silico simulation, a putative promoter region was apparently predicted between 1 kb upstream and 1 kb downstream of the start codon. In this region, +36T > C polymorphism was located on the GC box sequence in intron 1, and its functional effect on promoter activity was confirmed by luciferase reporter assay, introducing a new functional polymorphism of the GFPT1 gene. To examine its association with type 2 diabetes, we analyzed 2,763 Japanese (1,461 controls and 1,302 cases) and 330 Caucasians (190 controls and 140 cases). One possible association of +36T > C was observed in Caucasians, but no association of polymorphisms including +36T > C in intron 1 or haplotypes was observed in Japanese. Although we could not completely rule out a contribution to specific sub-groups or other populations, genetic variation of GFPT1 is unlikely to have a major role in the susceptibility to type 2 diabetes in Japanese.Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

The coat protein gene of grapevine leafroll associated closterovirus-3: cloning, nucleotide sequencing and expression in transgenic plants

Summary.  A lambda ZAP II cDNA library was constructed by cloning cDNA prepared from a high molecular weight double-stranded RNA (dsRNA, ca. 18 kb) isolated from grapevine leafroll associated closterovirus-3 (GLRaV-3) infected tissues. This cDNA library was immuno-screened with GLRaV-3 coat protein specific polyclonal and monoclonal antibodies and three immuno-positive clones were identified. Analysis of nucleotide sequences from these clones revealed an open reading frame (ORF) which was truncated at the end; the remainder of this ORF was obtained by sequencing a fourth clone that overlapped with one of the immunopositive clones. A total of 2028 bp was sequenced. The putative GLRaV-3 coat protein ORF, 939 bp, encodes a protein (referred to as p35) with a calculated of 34 866. Multiple alignment of the p35 amino acid sequence with coat protein sequences from other clostero-viruses revealed that the consensus amino acid residues (R and D) of filamentous plant viruses are preserved in the expected locations. The GLRaV-3 coat protein gene was then engineered for sense and antisense expression in transgenic plants. Transgenic Nicotiana benthamiana plants that contain the sense GLRaV-3 coat protein gene produced a 35 kDa protein that reacted with GLRaV-3 antibody in Western blot. Accepted January 10, 1997; Received November 12, 1996     

Characterization of Sm13, a tegumental antigen of Schistosoma mansoni

Sm13, a 13-kDa Schistosoma mansoni tegumental antigen, is one of the principal polypeptides recognized by antibodies from mice protectively vaccinated with adult-worm tegumental membranes. To obtain the complete gene encoding Sm13 we subcloned and sequenced a cDNA and a fragment of a genomic clone. The collated sequence contains 1088 nucleotides and represents the full-length open reading frame of the gene, encoding a protein of 104 amino acids with a calculated molecular mass of 11,923 Da, compatible with the native protein identified in the tegumental membranes. The sequence derived from genomic DNA contains a 45-nucleotide intron. The analysis of the predicted protein suggests the presence of both N- and C-terminal hydrophobic membrane-spanning segments, and the coding region contains no homology in the currently available data bases. Additionally, the coding region is preceded by putative CCAAT and TATA boxes that may be involved in the control of expression. Western-blot analysis and indirect immunofluorescence resulted in the identification of a 13-kDa protein (Sm13) in the tegument of adult worms. The present study reveals that Sm13 behaves as an integral membrane protein upon partitioning in Triton X-114 and that it is present in worms of 3 weeks or older but not in schistosomula or miracidia. Moreover, it is also specifically recognized by sera from some schistosomiasis patients in enzyme-linked immunosorbent assay and Western-blot analysis, suggesting that it is immunogenic in human schistosomiasis. Received: 17 February 2000 / Accepted: 22 March 2000     

Generation of maize cell lines containing autonomously replicating maize streak virus-based gene vectors

Summary.  Virion sense gene replacement derivatives of maize streak virus (MSV) were constructed with selectable marker expression cassettes based on the bialaphos resistance gene (bar) and the CaMV 35S promoter. The effect on replication of increasing the genomic size was tested by including: (1) the 550-bp maize adh I intron and 68-bp TMV Ω RNA leader sequences upstream of the bar genes; and (2) a fusion between the bar and E. coli glutathione reductase (gor) genes. Three recombinant viral vectors ranging in size from 2.7 kb to 4.8 kb replicated efficiently in biolistically transfected cells of suspension cultured Black Mexican sweetcorn (BMS) cells. Deletions greater than 39 bp 3′ of the stemloop sequence in the LIR adversely affected replicon release. Transformed bialaphos-resistant BMS cell lines were generated with all three vectors containing the bar gene: between 38 and 60% of cell lines contained replicating viral episomes. The replicons were structurally stable, replicated to copy numbers of over 500 per haploid genome, and were detected for more than one year after introduction. We noted significant enhancement of bar gene expression, both at the protein and RNA levels, associated with the presence of episomal vector DNA. The maize adhI intron and TMV Ω RNA leader sequences did not seem to have a significant effect on bar gene expression from replicating constructs, although expression from controls was enhanced. The results suggest that MSV-based constructs would provide a useful system for long-term gene amplification in cereal cell culture systems. Received September 4, 1998 Accepted March 4, 1999     

Genetic Variation in the <Emphasis Type="Italic">KIAA0319</Emphasis> 5′ Region as a Possible Contributor to Dyslexia

Reading disabilities (RD) have been linked and associated with markers on chromosome 6p with results from multiple independent samples pointing to KIAA0319 as a risk gene and specifically, the 5′ region of this gene. Here we focus genetic studies on a 2.3 kb region spanning the predicted promoter, the first untranslated exon, and part of the first intron, a region we identified as a region of open chromatin. Using DNA from probands with RD, we screened for genetic variants and tested select variants for association. We identified 17 DNA variants in this sample of probands, 16 of which were previously reported in public databases and one previously identified in a screen of this region. Based on the allele frequencies in the probands compared to public databases, and on possible functional consequences of the variation, we selected seven variants to test for association in a sample of families with RD, in addition to four variants which had been tested previously. We also tested two markers 5′ of this region that were previously reported as associated. The strongest evidence for association was observed with alleles of the microsatellite marker located in the first untranslated exon and haplotypes of that marker. These results support previous studies indicating the 5′ region of the KIAA0319 gene as the location of risk alleles contributing to RD.     

Characterisation of several heterogeneous species of defective RNAs derived from RNA 3 of cucumber mosaic virus

Summary. Preparations of double-stranded RNAs (dsRNAs) extracted from Nicotiana tabacum cv Xanthi plants infected with a subgroup IB isolate of Cucumber mosaic virus (CMV) were found to contain a heterogeneous population of defective RNAs (D-RNAs) derived from RNA 3. Characterised D-RNAs ranged in size from 1.5 to 1.9 kb and were derived either by a single in-frame deletion within the 3a or 3b genes or by means of double in-frame deletions within both genes. Also, northern blot hybridisation showed two other types of RNA derived from RNA 3: (a) RNA species of ca. 0.7 kb containing the 3′-terminus but lacking the 5′-terminus, which could be 3′-coterminal subgenomic of D-RNAs derived from the 3b gene and (b) RNA species of unknown origin of ca. 0.8 kb containing the 5′-terminus but lacking the 3′-terminus.     

5′ Regulatory and 3′ Untranslated Region Polymorphisms of Vitamin D Receptor Gene in South Indian HIV and HIV–TB Patients

Introduction  Vitamin D receptor (VDR) gene polymorphisms in the 5′ regulatory region (Cdx2 and A-1012G), coding region (FokI), and 3′ untranslated region (UTR; BsmI, ApaI, and TaqI) were studied to find out whether these polymorphisms are associated with susceptibility to or protection against HIV-1 and development of tuberculosis (TB) in human immunodeficiency virus (HIV)-1-infected patients. Study Subjects and Methods  The study was carried out in 131 HIV patients without TB (HIV+ TB−) and 113 HIV patients with TB (HIV+ TB+; includes 82 patients with pulmonary TB (HIV+ PTB+) and 31 with extra pulmonary TB), 108 HIV-negative pulmonary TB patients (HIV− PTB+), and 146 healthy controls. Results  Among the 5′ regulatory and coding region polymorphisms, significantly increased frequency of G/A genotype of Cdx-2 was observed in HIV+ TB− group compared to controls (p = 0.012, odds ratio (OR) 1.89 95% confidence interval (CI) 1.14–3.15). In the 3′ UTR genotypes, a decreased frequency of b/b genotype of BsmI in total HIV patients (p = 0.014, OR 0.54 95% CI 0.32–0.89) and increased frequencies of A/A genotype of ApaI in HIV+ TB+ patients (p = 0.041, OR 1.77 95% CI 1.02–3.06) and t/t genotype of TaqI in HIV+ PTB+ patients (p = 0.05, OR 2.32 95% CI 0.99–5.46) were observed compared to controls. Haplotype analysis revealed significantly increased frequencies of 3′ UTR haplotype B-A-t in HIV+ TB+ and HIV+ PTB+ groups (Pc = 0.030, OR 1.75 95% CI 1.14–2.66) and decreased frequencies of b-A-T haplotype in total HIV patients (Pc = 0.012, OR 0.46 95% CI 0.27–0.77), HIV+ TB− (p = 0.031 OR 0.48 95% CI 0.25–0.89), and HIV+ PTB+ groups (Pc = 0.04, OR 0.47 95% CI 0.23–0.89) compared to controls. Conclusions  The results suggest that VDR gene 3′ UTR haplotype b-A-T may be associated with protection against HIV infection while B-A-t haplotype might be associated with susceptibility to development of TB in HIV-1-infected patients.     

5′-UTR Structural Organization, Transcript Expression, and Mutational Analysis of the Human Rab Geranylgeranyl Transferase α-Subunit (RABGGTA) Gene

Hermansky-Pudlak syndrome (HPS) is a recessively inherited disease with dysfunction of several related subcellular organelles including platelet-dense granules, melanosomes, and lysosomes. Our recent identification of the mutation in murine Rab geranylgeranyl transferase α-subunit gene (Rabggta) in one mouse model of HPS, the gunmetal mouse, suggested that human patients with similar phenotypes might have mutations in the human orthologous RABGGTA gene. This prompted reanalysis of the 5′-untranslated structure of the human RABGGTA gene in normal individuals and in patients with deficiencies of platelet-dense granules (αδ-SPD), alpha granules (α-SPD or gray platelet syndrome, GPS) or alpha plus dense granules (αδ-SPD). We report the complete sequence of intron α of RABGGTA and demonstrate that exon α is immediately upstream of intron α. The exon/intron structural organization of the 5′-untranslated region (UTR) of human RABGGTA was found to be similar to that of the mouse Rabggta gene. However, exons α and introns α are not homologous between mouse and human. Features of the 5′-UTR of RABGGTA suggest it is a housekeeping gene. While obvious disease-causing mutations of human RABGGTA were not found in our existing SPD patients by sequencing its entire coding region, several polymorphisms of RABGGTA including a putative cryptic splicing mutation in intron 4 were identified. Knowledge of the 5′-UTR structure of RABGGTA and its common polymorphisms will be useful for mutation screening or linkage analysis in patients with albinism, thrombocytopenia, or platelet SPD.     

Non-homologous recombination between Alu and LINE-1 repeats caused a 430-kb deletion in the dystrophin gene: a novel source of genomic instability

Although large deletions in the dystrophin gene have been identified in more than two-thirds of Duchenne and Becker muscular dystrophy patients, the molecular mechanisms that lead to the generation of these deletions are largely unknown. Here, Alu and LINE-1 (L1) repetitive elements were shown to be present at one or other of the two ends, respectively, of a 430-kb deletion in the dystrophin gene. The breakpoint of the deletion, which stretches from exons 2 to 7, was defined more precisely by polymerase chain reaction (PCR) walking on introns 1 and 7. Finally, the region containing the breakpoint was amplified as a fragment of more than 10 kb. Sequencing of the deletion endpoint revealed the presence of an Alu sequence in intron 1, 25 kb downstream from the 3′ end of exon 1 that was joined directly to an L1 sequence in intron 7, 4.5 kb downstream from the 3′ end of exon 7. The deletion was calculated to be 430 kb. To our knowledge, this is a novel recombination event joining non-homologous Alu and L1 repeats, and is the largest known intrachromosomal deletion that is thought to involve repetitive genetic elements. Sequence characteristics around the breakpoint are discussed. Received: July 10, 2000 / Accepted: August 23, 2000     

Nucleotide sequence of a Dianthovirus RNA1-like RNA found in grassy stunt-diseased rice plants

Summary.  A Dianthovirus RNA1-like RNA (DR1L RNA, 4486 nucleotides in length) was found in grassy stunt-diseased rice plants together with Rice grassy stunt virus (RGSV). DR1L RNA has characteristics common to Dianthovirus RNA1 such as (1) presence of a GGAUUUU potential shifty-heptanucleotide at the end of the 5′-proximal ORF, which encodes a 35-kDa protein, followed by a 77-nucleotide sequence capable of forming a stem-loop structure for an efficient − 1 frameshift to express the downstream region in a 96-kDa putative replicase protein, (2) presence of nearly identical 17-nucleotide sequences in the 5′-terminal region and in a region upstream of an ORF encoding a 28-kDa, putative capsid protein (CP), and (3) near identity of the 3′-terminal 20 nucleotides to those of Dianthovirus RNAs. Western blot analysis using an antiserum against the C-terminal domain of the putative CP and RT-PCR analysis using primers specific to DR1L RNA of fractions after sucrose density gradient centrifugation of RGSV nucleoproteins indicated that DR1L RNA is associated with the 28-kDa putative CP but not with the 36-kDa RGSV CP. Two additional ORFs for 15-kDa and 33-kDa proteins were present in DR1L RNA although their expression in plants and functions are not known. Received May 15, 2000 Accepted August 24, 2000