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PubMed2006

Isolation of extremely AT-rich genomic DNA and analysis of genes encoding carbohydrate-degrading enzymes from Orpinomyces sp. strain PC-2.

An effective method for extraction of intact genomic DNA from the extremely AT-rich polycentric anaerobic fungus Orpinomyces sp. strain PC-2 has been developed. This procedure involves removal of glycogen-like storage polysaccharides using hexadecyltrimethylammonium bromide (CTAB) and high salt washes. The DNA was digested with various restriction enzymes and was suitable for use as a PCR template, for Southern blotting, and for genomic library construction. Genomic DNA analysis of three representative genes (celE, bgl1, and xynA) encoding (hemi-) cellulolytic enzymes of the fungus revealed multiplicity of family 5 endocellulase genes (celE-like), and family 1 beta-glucosidase genes (bgl1-like), but only a single copy of family 11 xylanase gene (xynA).

AT Rich Sequence↗

PubMed1991

Schistosoma japonicum: analysis of eggshell protein genes, their expression, and comparison with similar genes from other schistosomes.

As the egg of Schistosoma japonicum plays a central role in transmission and in pathogenesis, we sought to understand the molecular biology of egg formation. In this study we characterized an eggshell protein gene of S. japonicum and compared it with similar genes from S. mansoni and S. haematobium. To initiate studies on the eggshell protein genes of S. japonicum, a cloned genomic fragment containing an entire copy of a S. haematobium eggshell protein gene was used to identify three EcoRI hybridizing fragments of 2.6, 2.0, and 1.3 kbp in S. japonicum genomic DNA and to isolate three independent genomic clones from a S. japonicum genomic library. Two genomic clones, SJ 4-1 and SJ 3-1, contain at least two copies of the gene. The DNA sequence of a 2.0-kbp EcoRI fragment of clone SJ 3-1 showed two open reading frames (ORF), one of which showed a strong homology to the chorion proteins of insects. This ORF had 207 amino acids with a calculated molecular size of 18.5 kDa. The predicted peptide was glycine (50%) and tyrosine (10%) rich like other described schistosome eggshell proteins. Primer extension and the dideoxynucleotide sequence of the mRNA defined the cap site of the RNA and positioned the putative TATA and CAAAT elements and other cis-acting elements. Northern analysis demonstrated that eggshell protein mRNA was only detected in mature female parasites. The appearance of the female-specific mRNA was dependent on pairing with the male parasite and increased with egg production (as determined by hybridization intensity). A comparison of the DNA and deduced protein sequences of eggshell protein genes from S. japonicum with those of similar genes from S. mansoni and S. haematobium indicated that the genes are highly conserved, with S. mansoni and S. haematobium genes being more similar to each other than either is to S. japonicum.

Amino Acid Sequence↗

PubMed1994

Structural organisation of microsatellite families in the Leishmania genome and polymorphisms at two (CA)n loci.

In the present study, we have analysed the frequency and distribution of several microsatellite DNAs [(CA)n, (GGT)n and (GCA)n] in the genome of Leishmania. Hybridisation analysis on the molecular karyotypes of different Leishmania strains showed the presence of these three microsatellites on all chromosomes of the parasite. The number of microsatellite clusters appeared grossly similar among strains from different Old World complexes. However, these three microsatellite families showed an uneven distribution among heterologous chromosomes of the same strain. Moreover, restriction analysis of chromosome I in various strains of Leishmania infantum showed a strong clustering of these microsatellites in the same chromosomal region. A partial genomic library was screened with a (CA)n probe, and 21 positive clones were isolated. The sequencing of these clones confirmed the association of various microsatellites such as (CA)n, (CT)n, and (GCA)n. Finally, specific polymerase chain reaction amplification of two cloned (CA)n loci demonstrated allelic size polymorphisms among strains within L. infantum and Leishmania donovani. Most of the 34 strains analysed were found to be monoallelic, while two alleles were found in a small number of strains. The interest of these sequences for studies on ploidy and population genetics of the parasite is discussed.

Animals↗

PubMed1994

Organization of the mouse GP42/Basigin gene: a member of the Ig superfamily.

We have mapped and sequenced the GP42/Basigin gene isolated from a Balb/C mouse genomic library. The genomic organization and upstream, putative regulatory, regions of this gene have not been previously reported. Our data show that exon 5 of the GP42/Basigin gene encodes the carboxy proximal half of the second Ig-like domain, the highly conserved transmembrane region and a portion of the cytoplasmic tail. This inclusion of Ig-like and other functional domains in a single exon is unusual. Splice junction analysis indicates that two reported alternate GP42/Basigin cDNA isoforms are likely due to cloning artifacts. In addition, we find that GP42/Basigin is polymorphic in mice. Our data also support the proposal that the transmembrane domain and portions of the cytoplasmic region of GP42/Basigin have been evolutionarily conserved.

Amino Acid Sequence↗

PubMed2006

Molecular genetic transfection of the coccidian parasite Sarcocystis neurona.

Sarcocystis neurona is an apicomplexan parasite that is the major cause of equine protozoal myeloencephalitis (EPM). The biology of this pathogen remains poorly understood in part due to unavailability of molecular genetic tools. Hence, with an objective to develop DNA transfection capabilities for S. neurona, the 5' flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules beta-galactosidase (beta-gal) and yellow fluorescent protein (YFP) could be detected in electroporated S. neurona, thereby confirming the feasibility of transgene expression in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of Toxoplasma gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express beta-gal and YFP. As shown in this study, these transgenic clones can be useful for analyzing growth rate of parasites in vitro and for assessing drug sensitivities. More importantly, the DNA transfection methods described herein should greatly facilitate studies examining intracellular parasitism by this important coccidian pathogen.

5' Flanking Region↗

PubMed1992

Cloning and characterization of the human thyroid hormone receptor beta 1 gene promoter.

The promoter region of the human (h) thyroid hormone receptor (TR) beta 1 gene was isolated from a human placenta genomic library. Primer extension and S1 nuclease mapping confirmed a single transcriptional start site. DNA sequence analysis of the 5' upstream region revealed the existence of a putative thyroid response element (TRE) which is highly homologous to TREs found in several thyroid hormone responsive genes. Binding of hTR protein to the promoter region of the hTR beta 1 gene was confirmed by gel mobility shift assay. A transient transfection study demonstrated that hTR activated the expression of a reporter gene containing the promoter sequence of the hTR beta 1 gene in a hormone dependent manner. The TRE in the hTR beta 1 gene promoter may be involved in the autoregulation of hTR beta 1 gene expression.

Base Sequence↗

PubMed1999

A novel subgroup of class I G-protein-coupled receptors.

Based on structural similarities of an expressed sequence tag with the platelet-activating factor (PAF) receptor a cDNA clone encoding a novel G-protein-coupled receptor (GPCR), named GPR34, was isolated from a human fetal brain cDNA library. Genomic DNA analyses revealed the receptor to be encoded by an intronless single-copy gene at Xp11. 3-11.4. The predicted 381-amino-acid protein disclosed all structural features characteristic of a member of the class I GPCR family. Except an obvious sequence homology in transmembrane domain 6, no further similarities to the PAF receptor or any other known GPCR were found. The corresponding mouse receptor DNA was isolated from a genomic P1 library displaying a 90% amino acid identity compared to the human receptor. Phylogenetic studies showed that GPR34 is preserved among vertebrates, and the existence of GPR34 subtypes was demonstrated. The receptor mRNA is abundantly expressed in human and mouse tissues. In addition to the major 2-kb transcript, a 4-kb transcript was found only in mouse liver and testis. Expression of the human GPR34 in COS-7 cells followed by Western blot studies revealed specific bands of a highly glycosylated protein between 75 and 90 kDa. A number of potential ligands including phospholipids, leukotrienes, hydroxy-eicosatetraenoic acids, nucleotides and peptides were tested in functional assays. Since none of the applied substances led to significant changes in second messenger levels (cAMP and inositol phosphates), the natural ligand and coupling profile of this novel GPCR subgroup remains unknown.

Amino Acid Sequence↗

PubMed2001

Characterization of the antiapoptotic (p35) gene homologue of Spodoptera litura nucleopolyhedrosis virus (SlNPV).

Antiapoptotic genes of baculoviruses have been shown to prevent virus induced apoptosis in insect cells. Dot blot and Southern hybridizations of EcoRI genomic library and genomic digests of Spodoptera litura nucleopolyhedrosis virus (SlNPV) respectively give strong hybridization signals with antiapoptotic DNA (p35 gene) probe of the prototype Autographa californica nucleopolyhedrosis virus (AcNPV). Both the hybridizations indicate the presence of a homologous gene in the 1.8 kb EcoRI-Y fragment of SlNPV. The sequence of 1.244 kb region of this fragment encompasses an open reading frame coding for a polypeptide of 296 amino acids under sequential early (TATA) and late (TAAG) promoter motifs like that in other baculovirus p35 genes. The putative SlNPV p35 ORF expresses abundantly as a 35 kDa protein in Spodopterafrugiperda (Sf9) cells when allowed to express under the polyhedrin promoter of AcNPV.

Amino Acid Sequence↗

PubMed1986

Human Cu/Zn superoxide dismutase gene family: molecular structure and characterization of four Cu/Zn superoxide dismutase-related pseudogenes.

Four distinct human Cu/Zn superoxide dismutase (SOD1; EC 1.15.1.1)-related sequences were isolated from genomic DNA libraries. Genomic blots, heteroduplex analyses, and DNA sequencing showed that they are processed pseudogenes not residing on chromosome 21. Three of them originated from the 0.7-kilobase SOD1 mRNA, while the fourth was derived from the 0.9-kilobase mRNA species. Comparison between the coding sequences of the functional gene and two of the processed genes suggested that they integrated into the genome about 25 million years ago.

Animals↗

PubMed1989

Snake acetylcholine receptor: cloning of the domain containing the four extracellular cysteines of the alpha subunit.

The acetylcholine receptor (AcChoR) at the neuromuscular junction of elapid snakes binds cholinergic ligands but unlike other muscle AcChoRs does not bind alpha-bungarotoxin. Numerous studies indicate that the ligand-binding site of the AcChoR includes cysteine residues at positions 192 and 193 of the alpha subunit. We have previously shown that a synthetic dodecapeptide corresponding to residues 185-196 of the Torpedo AcChoR alpha subunit contains the essential elements of the ligand-binding site. In an attempt to elucidate the structural basis for the precise binding properties of snake AcChoR, we sequenced a portion of the snake AcChoR alpha subunit. First, a mouse AcChoR alpha-subunit cDNA probe was used to screen a size-selected snake (Natrix tessellata) genomic library. A genomic clone was isolated and was found to contain sequences homologous to the exon including the first two cysteines (Cys-128 and -142) of AcChoR alpha subunit. The domain of the alpha subunit from Natrix and cobra AcChoR (amino acid residues 119-222), which contains the four extracellular cysteines (128, 142, 192, and 193), was amplified by reverse transcription of mRNA and the polymerase chain reaction and then sequenced. The deduced amino acid sequence showed that the snake alpha subunit contains the two tandem cysteines at positions 192 and 193, resembling all other AcChoR alpha subunits. Sequence comparison revealed that the cloned region of the snake alpha subunit is highly homologous (75-80%) to other muscle AcChoRs and not to neuronal AcChoR, which also does not bind alpha-bungarotoxin. In the presumed ligand-binding site, in the vicinity of Cys-192 and Cys-193, four major substitutions occur in the snake sequence--at positions 184 (Trp----Phe), 185 (Lys----Trp), 187 (Trp----Ser), and 194 (Pro----Leu). In addition, Asn-189 is a putative N-glycosylation site, present only in the snake. These changes, or part of them, may explain the lack of alpha-bungarotoxin-binding to snake AcChoR.

Amino Acid Sequence↗

PubMed2005

Identification of a gene involved in polysaccharide export as a transcription target of FruA, an essential factor for Myxococcus xanthus development.

Fruiting body development in Myxococcus xanthus is a multicellular event that is coordinated by exchanging intercellular signals. FruA is a transcription factor essential for fruiting body development and is thought to play a key role in the C-signal pathway. Here we present the first identification of a gene regulated by FruA. The gene was isolated from a genomic library via in vitro selection in a DNA binding assay by using the DNA-binding domain of FruA tagged with His(8) at the C-terminal end (FruA-DBD-H(8)). The gene, named fdgA (FruA-dependent gene A), encodes a protein homologous to the outer-membrane auxiliary family protein involved in the polysaccharide export system. FruA-DBD-H(8) bound the upstream promoter region of the fdgA gene from nucleotide -89 to nucleotide -64 with respect to the transcription initiation site, which was required for the induction of fdgA expression during development. fdgA mRNA induced during development was absent in a fruA deletion strain. The deletion of fdgA resulted in defective fruiting body formation and reduced sporulation efficiency (1% that of the parent strain). Moreover, FruA was required for the developmental expression of sasA, which is also involved in the biosynthesis of the lipopolysaccharide O-antigen and is required for fruiting body development. Furthermore, the expression of both fdgA and sasA was partially dependent on the C-signal. These findings expand our understanding of the signal transduction pathway mediated by FruA during development in M. xanthus.

Amino Acid Sequence↗

PubMed1987

Progress towards construction of a total restriction fragment map of a human chromosome.

We present an approach to the construction of an overlapping restriction fragment map of a single human chromosome. A genomic cosmid library genome was constructed from a mouse-human hybrid cell line containing chromosome 17 as its only human genetic component. Cosmids containing human inserts were isolated by hybridisation to a human Alu sequence. DNAs from ninety-six randomly chosen cosmids were digested with either EcoRI or HindIII, end-labelled with 35S-dATP and analysed using agarose gel electrophoresis. Comparison of the restriction fragment patterns revealed two pairs of overlapping clones, that were confirmed by cross-hybridization of the overlapping fragments. The two pairs of cosmids both mapped to human chromosome 17, as shown by hybridization to a panel of somatic cell hybrids. These data demonstrate that the generation of an overlapping cosmid map along a human chromosome is feasible, representing an intermediate step towards the complete sequencing of a human chromosome.

Animals↗

PubMed1989

Cloning and characterization of bovine cytochrome P-450(11 beta) genes.

We isolated 4 different clones of the P-450(11 beta) gene from a bovine genomic library. These genomic clones were highly homologous with each other. Two of the isolated clones were pseudogenes. Determination of its nucleotide sequences indicated that the bovine P-450(11 beta) gene is divided into 9 exons by 8 introns and that it is about 8.5 kb in total length. The number of exons and the locations of intron insertion into the P-450(11 beta) gene are identical with those in the case of P-450(SCC), but different from those of other microsomal P-450s.

Animals↗

PubMed1992

Characterization of swine short interspersed repetitive sequences.

Swine genomic DNA segments containing repetitive sequences were isolated from a porcine genomic library using genomic DNA as a probe. Three fragments containing the repetitive sequences from two of the primary phage clones were subcloned for sequence analysis, which revealed six new PRE-1 repetitive families other than those reported earlier by Singer et al. (Nucleic Acids Research 15, 2780, 1987). The frequency of the repetitive sequences in the swine genome was estimated at 2 x 10(6) per diploid genome. Sequence analysis revealed similarities between these repetitive sequences and that of arginine-tRNA gene.

Animals↗

PubMed1994

Genomic cloning and localization by FISH and linkage analysis of the human gene encoding the primary subunit NMDAR1 (GRIN1) of the NMDA receptor channel.

A cDNA clone of the NMDAR1 (isoform E) has been used to screen both lambda and cosmid genomic libraries. A genomic phage clone was identified and sequenced and was found to contain some of the 3' coding regions of the GRIN1 gene. This clone was used to localize the gene using fluorescent in situ hybridization (FISH) to normal chromosomes, and also to a lymphoblastoid cell line containing a translocation involving chromosomes 9 and 15. FISH localized the gene to chromosome 9q34.3. The clone was used to screen a panel of genomic DNAs cut with 20 restriction enzymes. A VNTR sequence 5' to the gene, which was polymorphic for a number of restriction enzymes, was detected. A PvuII fragment of the genomic clone was found to detect the VNTR on Southern hybridization. The polymorphic VNTR marker was mapped against chromosome 9q34 markers using linkage analysis in the CEPH families. The GRIN1 gene was linked to D9S7 with a maximum lod score of 20.09 at zero recombination fraction in males and 0.03% recombination in females.

Alleles↗

PubMed2004

An ste20 homologue in Ustilago maydis plays a role in mating and pathogenicity.

The mitogen-activated protein kinase (MAPK) pathways are conserved from fungi to humans and have been shown to play important roles in mating and filamentous growth for both Saccharomyces cerevisiae and dimorphic fungi and in infectivity for pathogenic fungi. STE20 encodes a protein kinase of the p21-activated protein kinase family that regulates more than one of these cascades in yeasts. We hypothesized that an Ste20p homologue would play a similar role in the dimorphic plant pathogen Ustilago maydis. The full-length copy of the U. maydis gene was obtained from a genomic library; it lacked introns and was predicted to encode a protein of 826 amino acids, whose sequence confirmed its identity as the first Ste20p homologue to be isolated from a plant pathogen. The predicted protein contained both an N-terminal regulatory Cdc42-Rac interactive binding domain and a C-terminal catalytic kinase domain. Disruption of the gene smu1 resulted in a delayed mating response in a mating-type-specific manner and also in a severe reduction in disease production on maize. Unlike the Ustilago bypass of cyclase (ubc) mutations previously identified in genes in the pheromone-responsive MAPK cascade, mutation of smu1 does not by itself act as an extragenic suppressor of the filamentous phenotype of a uac1 mutant. Thus, the direct connection of Smu1p to MAPK cascade function has yet to be established. Even so, Smu1, though not absolutely required for mating, is necessary for wild-type mating and pathogenicity.

Blotting, Northern↗

PubMed1992

Development of a polymerase chain reaction-based diagnosis of Trichomonas vaginalis.

We developed a polymerase chain reaction (PCR)-based test for detecting the protozoan parasite Trichomonas vaginalis. Genomic libraries were constructed from two independent clinical isolates of T. vaginalis. From these libraries, 12 genomic clones were purified, sequenced, and then screened for uniqueness by computer-assisted sequence comparisons. PCR reactions were performed to evaluate eight PCR-primer pairs, including a primer pair that targeted the T. vaginalis ferredoxin gene. All eight primer pairs yielded PCR products of the expected sizes. However, six of the primer pairs amplified their respective target sequences in limited numbers of clinical T. vaginalis isolates, suggesting the presence of significant genomic variability among isolates. An exception was a primer pair, termed TVA5-TVA6, that amplified a 102-bp genomic sequence, termed A6p, in all of 24 clinical isolates. The A6p sequence was not detected by PCR in human DNA or in a wide variety of flagellates, ciliates, or bacteria tested. The A6p sequence appears highly selective for a broad range of T. vaginalis isolates and holds promise for PCR-based diagnosis of the parasite.

Animals↗

PubMed1987

Structure of two developmentally regulated Dictyostelium discoideum ubiquitin genes.

A previously isolated cDNA clone, pLK229, that is specific for mRNA developmentally expressed during Dictyostelium discoideum spore germination and multicellular development, was used to screen two genomic libraries. Two genomic sequences homologous to pLK229 were isolated and sequenced. Genomic clone p229 is identical to the cDNA clone pLK229 and codes for a polypeptide of 381 amino acids. This polypeptide is composed of five tandem repeats of the same 76-amino-acid sequence. Clone lambda 229 codes for a protein of 229 amino acids, containing three tandem repeats of the identical 76-amino-acid sequence. A computer search for homology to known proteins revealed that the 76-amino-acid repeat was identical to human and bovine ubiquitin except for two amino acid differences.

Amino Acid Sequence↗