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PubMed2001

[Cloning and phylogenetic analysis of hepatitis-associated TT virus subgenome].

Polymerase chain reaction (PCR) was utilized for the DNA amplification from transfusion transmitted virus (TTV) positive serum samples. Five TTV DNA fragments, overlapped about 90% of the genome, were amplified by long template PCR for the generation of TTV subgenome. Recombinant plasmids were obtained by directly inserting PCR products into pT-Adv vector, and DNA sequence analyses showed they were TTV DNA fragments. By using specific restriction enzymes, five TTV DNA fragments were ligated into a TTV DNA subgenome clone and named as TTV021. TTV021 has been deposited in GenBank database with the accession number AF254410. The results of computer analyses showed that TTV021, 3472 nt long, contains two open reading frames (ORF1, 785 aa; ORF2, 146 aa). Identity alignments between TTV021 and other TTV isolates indicated several high conserved regions existed. Phylogenetic analysis of 356 nt from TTV021 suggested that the isolate has close evolutionary relationship with CHN1 (type 1a), but has far relation with other TTV isolates.

Circoviridae↗
PubMed1999

New mutations, polymorphisms, and rare variants in the ATM gene detected by a novel SSCP strategy.

The gene for ataxia-telangiectasia, ATM, spans about 150 kb of genomic DNA. ATM mutations are found along the entire gene, with no evidence of a mutational hot spot. Using DNA as the starting material, we screened the ATM gene in 92 A-T patients, using an optimized single-strand conformation polymorphism (SSCP) technique that detected all previously known mutations in the polymerase chain reaction (PCR) segments being analyzed. To expedite screening, we sequentially loaded the SSCP gels with three different sets of PCR products that were pretested to avoid overlapping patterns. Many of the DNA changes we detected were intragenic polymorphisms. Of an expected 177 unknown mutations, we detected approximately 70%, mostly protein truncating mutations (that would have been detectable by protein truncation testing if RNA starting material had been available). Mutations have now been defined for every exon of the ATM gene. Herein, we present 35 new mutations and 34 new intragenic polymorphisms or rare variants within the ATM gene. This is the most comprehensive compilation of ATM polymorphisms assembled to date. Defining polymorphic sites as well as mutations in the ATM gene will be of great importance in designing automated methods for detecting mutations.

Ataxia Telangiectasia↗
PubMed1994

The chondroitin sulfate proteoglycan NG2 is a tumour-specific antigen on the chemically induced rat chondrosarcoma HSN.

N-terminal sequences of 19 and 11 amino acids obtained from 2 different tryptic fragments of the tumour-specific antigen on the chemically induced rat chondrosarcoma HSN show a 100% homology with the rat chondroitin sulfate proteoglycan NG2. Using a scheme of overlapping oligonucleotide primers we have cloned by PCR amplification the cDNA for the specific antigen of the HSN tumour that is immunogenic in immunocompetent CBH/Cbi rats and now report that its cDNA sequence is identical to that of NG2. The cDNA codes for a transmembrane protein of 2,325 amino acids with a large extracellular domain of 2,224 amino acids containing 2 cysteine-rich regions, a transmembrane domain (25 amino acids) and a short cytoplasmic tail (76 amino acids). The tumour-specific determinant was found to lie between amino acid residues 556 and 992 on the core glycoprotein.

Amino Acid Sequence↗
PubMed1994

Nucleotide sequence analysis of puma lentivirus (PLV-14): genomic organization and relationship to other lentiviruses.

The complete nucleotide sequence of an isolate of puma lentivirus (PLV-14) was obtained by an inverse polymerase chain reaction (I-PCR) technique and confirmed by conventional PCR. Both methods were used to amplify overlapping regions of proviral DNA, for cloning and sequencing, from genomic DNA isolated from PLV-14 infected Florida puma (Felis concolor coryi) peripheral blood mononuclear cells (PBMC). The provirus has a total length of 9100 nucleotides and the genomic organization of presumed protein coding regions are similar to those seen in other members of the lentivirus family, i.e., three large open reading frames gag, pol, and env as well as smaller intergenic regions that apparently encode regulatory proteins vif and 3' rev by positional and sequence similarity to those seen in other lentiviruses. Two additional open reading frames were identified in the env region and their function (if any) is unknown. The length of the PLV-14 long terminal repeat (LTR) was found to be shorter than the LTRs of feline immunodeficiency virus (FIV). The sequence homology between PLV-14 and other lentiviruses demonstrates that PLV-14 is most closely related to FIV from domestic cats. However, the extent of sequence divergence of each retroviral gene segment is large (e.g., percentage sequence similarity between FIV and PLV-14 env is 8% amino acid and 37% nucleotide similarity), indicating relatively ancient divergence of these feline lentiviral genomes.

Amino Acid Sequence↗
PubMed1994

Molecular epidemiology of Tay-Sachs disease in Europe.

The abnormalities in the gene coding for the beta-hexosaminidase alpha subunit were analysed from fibroblast's RNAs of 42 Tay-Sachs patients (seven with adult or late onset of Tay-Sachs disease and 35 with infantile Tay-Sachs disease). After first strand synthesis by random priming, PCR was used to amplify in two overlapping fragments (868 and 949 bp) the entire coding region. These amplified products were first studied for changes in size by agarose gel electrophoresis to screen for splicing mutations leading to exon skipping or cryptic splice site activation. For each patient, the two overlapping cDNA fragments were subjected to chemical mismatch cleavage analysis using hydroxylamine to modify C-containing mismatches and osmium tetroxide to modify T-containing mismatches. DGGE was used to screen for mutations in the coding region spanning exon 2 to exon 6, a region putatively encompassing the active site and therefore a potential hot spot of mutations associated with Tay-Sachs disease. To increase the sensitivity of the technique, a 30 bp GC-clamp has been added at the 5' end of the sense oligonucleotide to amplify a fragment of 629 bp. The computerized analysis found that single base changes in domain spanning from nt 313 to nt 693 can be distinguished. Fragments displaying an altered melting behavior or a cleaved product were further analysed by direct sequencing of the amplified material. These methods as a whole allowed us to identify 30/38 alleles studied (79%) with 15 point mutations and one 4 bp insertion detected.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed2005

Cloning, distribution and up-regulation of the teleost fish MHC class II alpha suggests a role for granulocytes as antigen-presenting cells.

The major histocompatibility complex (MHC) class II alpha chain gene of the teleost fish gilthead seabream (Sparus aurata), Spau-DAA, has been characterized. We cloned, sequenced and studied its polymorphism, before evaluating its expression in resting seabream leucocytes, tissues and tumor cells as well as in primed leucocytes. A complete allele was obtained by overlapping sequence fragments obtained by RT-PCR. The full-length Spau-DAA*101 comprises 1840 bp with a 5'-UTR region of 84 bp, an ORF of 729 bp and a 3'-UTR of 1027 bp. The putative protein of 242 residues shows homology with known MHC class II alpha genes, varying from 71 to 28% in other fish and humans, respectively. The protein sequence showed all the important features: leader peptide, alpha1, alpha2 and CP/TM/CYT regions, conserved cysteines and N-glycosylation site. The phylogenetic tree shows that it is included in the cluster containing the Percomorpha subclass and far from the human and shark genes. It is polymorphic, as seen when we sequenced the complete ORF of 11 alleles showing most of the amino acidic changes in the alpha1 domain, where the peptide-binding region (PBR) is found. Spau-DAA mRNA expression was mainly found in peritoneal exudate leucocytes, head-kidney, spleen, thymus and gill. Minor expression was detected in gut, brain, liver and PBLs. RT-PCR expression studies in isolated leucocyte subpopulations revealed, for the first time in the literature, that acidophilic granulocytes show high MHC class II gene expression. Apart from these granulocytes lymphocytes also express the Spau-DAA gene, although other cell types may also do the same. Finally, incubation of head-kidney leucocytes with yeast cells or pathogenic bacteria up-regulates Spau-DAA gene expression whilst incubation with ConA, ConA+LPS or PHA does not. The possible involvement of the seabream MHC class II alpha gene in the fish defence and antigen presentation are discussed.

Amino Acid Sequence↗
PubMed1998

Production of rat salivary cystatin S variant polypeptides in Escherichia coli.

Cystatins are protein inhibitors of papain and related cysteine proteinases. A series of continuous synthetic peptides corresponding to the entire sequence of rat salivary cystatin was used to localize the binding domains of the cystatin to papain. Several synthetic peptides, one from the aminoterminal sequence (peptide 1-24) and others from the carboxylterminal (peptides 66-79, 66-90, 79-90, 79-114), showed binding to papain, but none of the peptides showed inhibition of papain activity. Three recombinant rat salivary cystatin variants (N-terminal truncated protein lacking amino acid residues 1-9; variant 49-53, in which amino acid residues QVVAG of rat salivary cystatin had been replaced with amino acid residues LVL in mutant protein; and variant 65-78, in which amino acid residues 65-78 had been replaced with amino acids PG in mutant protein) were produced using the Escherichia coli expression system pGex-4T. To generate N-terminal truncated protein the desired coding region of the cystatin gene was amplified by polymerase chain reaction (PCR). To produce the variants 49-53 and 65-78, a PCR-based approach of gene splicing by overlap extension was used. Recombinant cystatin proteins were produced as insoluble inclusion bodies as fusion proteins with a glutathione S-transferase (GST) carrier. After solubilization with urea the GST carrier was cleaved from the fusion protein with thrombin and cystatin variants purified by fast liquid chromatography on a MonoQ column. The purified proteins reacted with antibodies to rat salivary cystatin. The N-terminal truncated and variant 49-53 exhibited very little inhibitory activity towards papain, whereas variant 65-78 exhibited papain-inhibitory activity similar to the full-length recombinant cystatin.

Alternative Splicing↗
PubMedDate not supplied

Expression of an inducible nitric oxide synthase gene in rainbow trout Oncorhynchus mykiss.

Using oligonucleotide primers based on mammalian nitric oxide synthases (NOS), expression of an inducible NOS (iNOS) gene was detected in head kidney and gill tissue of bacterially-challenged rainbow trout. Three overlapping fragments were amplified by RT-PCR and used to construct a contiguous sequence of 1410bp, with high nucleotide homology to iNOS in birds (61%) and mammals (57-59%). The nucleotide sequence translated in one reading frame to produce a partial peptide containing 470 amino acids, with 69-71% amino acid homology with mammalian iNOS, 81% homology with chicken iNOS and 85% homology with a partial (492bp) goldfish iNOS sequence. In vitro stimulation of head kidney macrophages with LPS also induced expression of the trout iNOS RNA, with optimal expression seen using 20-50 microg/ml LPS at 2h to 6h post-stimulation. The evolutionary and functional significance of the trout iNOS sequence are discussed.

Amino Acid Sequence↗
PubMed1999

A signal recognition particle receptor gene from the early-diverging eukaryote, Giardia lamblia.

The molecular mechanisms for targeting and translocation of secreted proteins are highly conserved from bacteria to mammalian cells, although the machinery is more complex in higher eukaryotes. To investigate protein transport in the early-diverging eukaryote, Giardia lamblia, we cloned the gene encoding the alpha subunit (SRalpha) of the signal recognition particle (SRP) receptor. SRalpha is a small GTPase that functions in SRP-ribosome targeting to the ER. Sequence and phylogenetic analyses showed that SRalpha from G. lamblia is most homologous to SRalpha proteins from higher eukaryotes, although it lacks some conserved motifs. Specifically, giardial SRalpha has an N-terminal extension that enables SRalpha of higher eukaryotes to interact with a beta subunit that anchors it in the ER membrane. While the C-terminal regions are similar, giardial SRalpha lacks a prominent 13 amino acid regulatory loop that is characteristic of higher eukaryotic versions. Thus, giardial SRalpha resembles that of higher eukaryotes, but likely diverged before the advent of the regulatory loop. The 1.8 kb SRalpha transcript has extremely short untranslated regions (UTRs): a 1-2 nt 5'- and a 9 nt 3' UTR with the polyadenylation signal overlapping with the stop codon. RT-PCR, Northern and Western analyses showed that SRalpha is present at relatively constant levels during vegetative growth and encystation, even though there are extensive changes in endomembrane structures and secretory activity during encystation. Imnuno-EM showed that SRalpha localizes to ER-like structures, strengthening the observation of a typical ER in G. lamlia. Unexpectedly, SRalpha was also found in the lysosome-like peripheral vacuoles, suggesting unusual protein traffic in this early eukaryote. Our results indicate that the eukaryotic type of cotranslational transport appeared early in the evolution of the eukaryotic cell.

Amino Acid Sequence↗
PubMed1996

Cloning of a third mammalian Na+-Ca2+ exchanger, NCX3.

NCX3 is the third isoform of a mammalian Na+-Ca2+ exchanger to be cloned. NCX3 was identified from rat brain cDNA by polymerase chain reaction (PCR) using degenerate primers derived from the sequences of two conserved regions of NCX1 and NCX2. The NCX3 PCR product was used to isolate two overlapping clones totalling 4.8 kilobases (kb) from a rat brain cDNA library. The overlapping clones were sequenced and joined at a unique Bsp106I restriction enzyme site to form a full-length cDNA clone. The NCX3 cDNA clone has an open reading frame of 2.8 kb encoding a protein of 927 amino acids. At the amino acid level, NCX3 shares 73% identity with NCX1 and 75% identity with NCX2 and is predicted to share the same membrane topology as NCX1 and NCX2. Following addition of a poly(A)+ tail to the NCX3 clone, exchanger activity could be expressed in Xenopus oocytes. NCX3 was also expressed in the mammalian BHK cell line. NCX3 transcripts are 6 kb in size and are highly restricted to brain and skeletal muscle. Linkage analysis in the mouse indicated that the NCX family of genes is dispersed, since the NCX1, NCX2, and NCX3 genes mapped to mouse chromosomes 17, 7, and 12, respectively.

Amino Acid Sequence↗
PubMed2005

Structural insights into sulfite oxidase deficiency.

Sulfite oxidase deficiency is a lethal genetic disease that results from defects either in the genes encoding proteins involved in molybdenum cofactor biosynthesis or in the sulfite oxidase gene itself. Several point mutations in the sulfite oxidase gene have been identified from patients suffering from this disease worldwide. Although detailed biochemical analyses have been carried out on these mutations, no structural data could be obtained because of problems in crystallizing recombinant human and rat sulfite oxidases and the failure to clone the chicken sulfite oxidase gene. We synthesized the gene for chicken sulfite oxidase de novo, working backward from the amino acid sequence of the native chicken liver enzyme by PCR amplification of a series of 72 overlapping primers. The recombinant protein displayed the characteristic absorption spectrum of sulfite oxidase and exhibited steady state and rapid kinetic parameters comparable with those of the tissue-derived enzyme. We solved the crystal structures of the wild type and the sulfite oxidase deficiency-causing R138Q (R160Q in humans) variant of recombinant chicken sulfite oxidase in the resting and sulfate-bound forms. Significant alterations in the substrate-binding pocket were detected in the structure of the mutant, and a comparison between the wild type and mutant protein revealed that the active site residue Arg-450 adopts different conformations in the presence and absence of bound sulfate. The size of the binding pocket is thereby considerably reduced, and its position relative to the cofactor is shifted, causing an increase in the distance of the sulfur atom of the bound sulfate to the molybdenum.

Amino Acid Sequence↗
PubMed1997

Dystrophin point mutation screening using a multiplexed protein truncation test.

We report here the first use of a multiplexed protein truncation test for the high throughput screening of dystrophin point mutations. We have developed a substantially more robust and efficient procedure incorporating large savings in cost which uses muscle biopsy or lymphocyte total RNA as the template. The entire dystrophin open reading frame is screened in only five overlapping fragments using a long RT-PCR strategy to amplify dystrophin cDNA in excess of 3.7 kb. These five fragments are uniquely transcribed and translated in vitro in a single multiplexed reaction containing magnesium ions to reduce nonspecific internal initiation of translation. We have used this system to analyze mutations in 11 Duchenne muscular dystrophy patients (10 unrelated) with previously uncharacterized mutations. A single truncating mutation was identified in all patients, which was confirmed at the genomic level. Multiplex PTT provides the most efficient method for point mutation screening in this large gene and has potential applications to several disease genes with a significant proportion of truncating mutations.

Amino Acid Sequence↗
PubMed2005

Variation in selected regions of blaTEM genes and promoters in Haemophilus influenzae.

OBJECTIVES: To determine if the TEM beta-lactamases of Haemophilus influenzae are TEM-1 or derivatives thereof and associated exclusively with the overlapping Pa/Pb promoters. METHODS: Single nucleotide specific PCR was used to discriminate the polymorphic nucleotides at positions 32 and 317 of the bla(TEM) genes of a collection of TEM-positive strains. RESULTS: All bla(TEM) genes were found to be bla(TEM-1) or derivatives thereof and none bla(TEM-2). The bla(TEM) genes were associated with the P3 promoter, the Pa/Pb promoters or a novel promoter produced as a result of a 135 bp deletion and a G162T substitution. CONCLUSIONS: The genetic features of bla(TEM) genes in H. influenzae are different from those in Enterobacteriaceae and more variable than previously recognized.

Amino Acid Substitution↗
PubMed1998

Nucleotide sequence, genetic organization and expression strategy of the double-stranded RNA associated with the '447' cytoplasmic male sterility trait in Vicia faba.

The entire nucleotide sequence of the double-stranded (ds) RNA associated with the unconventional '447' cytoplasmic male sterility (CMS) trait in Vicia faba was determined from overlapping cDNA clones and by RT-PCR. Confirming previous observations, it was found that the negative-strand was continuous and 17,635 nt long, while the positive-strand featured an interruption, probably a nick, that could potentially define two subgenomic RNAs of 2735 nt and 14,900 nt, with the smaller RNA being located on the 5' side. The entire positive-strand could encode a single in-frame ORF starting at the first AUG at position 42-44 and ending with a TGA at 17,517-17,519. This long potential polypeptide with a predicted molecular mass of 654,109 is the largest described to date in the plant kingdom and contains conserved amino acid sequence motifs typical of viral helicases and RNA-dependent RNA polymerases (RDRP). Only limited sequence homology was detected with the ORF B encoded by the hypovirulence-associated dsRNA of chestnut blight fungus, a dsRNA replicon similarly contained in host-derived membranous vesicles and considered to share a common ancestry with potyviruses. By contrast, the helicase and RDRP domains were in the same respective arrangement and shared extensive sequence homologies with those identified in the polyprotein encoded by the dsRNA isolated from Japonica rice, another dsRNA replicon featuring a specific nick in the positive-strand. Although no proteolytic self-cleavage activity has yet been demonstrated, it appears likely that this long ORF is a polyprotein that undergoes proteolytic maturation, with one of the polypeptides derived by self-cleavage being the determinant of the CMS trait.

Amino Acid Sequence↗
PubMed2005

Genomic RNA sequence of Feline coronavirus strain FIPV WSU-79/1146.

A consensus sequence of the Feline coronavirus (FCoV) (strain FIPV WSU-79/1146) genome was determined from overlapping cDNA fragments produced by RT-PCR amplification of viral RNA. The genome was found to be 29 125 nt in length, excluding the poly(A) tail. Analysis of the sequence identified conserved open reading frames and revealed an overall genome organization similar to that of other coronaviruses. The genomic RNA was analysed for putative cis-acting elements and the pattern of subgenomic mRNA synthesis was analysed by Northern blotting. Comparative sequence analysis of the predicted FCoV proteins identified 16 replicase proteins (nsp1-nsp16) and four structural proteins (spike, membrane, envelope and nucleocapsid). Two mRNAs encoding putative accessory proteins were also detected. Phylogenetic analyses confirmed that FIPV WSU-79/1146 belongs to the coronavirus subgroup G1-1. These results confirm and extend previous findings from partial sequence analysis of FCoV genomes.

Coronavirus, Feline↗
PubMed1996

cDNA cloning of mouse nebulin. Evidence that the nebulin-coding sequence is highly conserved among vertebrates.

Nebulin is a family of giant myofibrillar proteins with molecular masses ranging over 700-900 kDa. Using a human nebulin cDNA probe, we isolated three nebulin cDNA clones from a mouse skeletal muscle cDNA library. These three clones, labeled 8c. 7a and 4b. carry inserts of 2.0, 3.0 and 3.5 kb, respectively. In Northern blots, each insert detected the same approximately = 25 kb message from skeletal muscle as the human nebulin probe, while detecting no messages from cardiac muscle. Sequence data in combination with reverse-transcriptase PCR indicates that clones 7a and 8c overlap to form 4076 bp contiguous sequence. Alignment with the published full-length human nebulin sequence indicates that clone 4b overlaps with clone 7a over 1596 bp. However, after the first 798-bp overlap, the sequence of these two mouse nebulin clones diverge, suggesting that they derive from distinct transcripts encoding isoforms of mouse nebulin. The mouse nebulin clones encode a series of = 245-residue super repeats, each of which can be subdivided into seven = 35-residue, weakly repeating modules centered around a conserved tyrosine residue, consistent with the human nebulin sequence. The mouse nebulin clones align along the central third of the full-length human sequence, corresponding to super repeats 8-16 of the 22 super repeats found in human nebulin. The translated sequence is greater than 90% identical to the human sequence, with the exception of a 200-amino-acid region at the C-terminus of clone 4b, which is less than 60% identical. In genomic Southern blots, a mouse nebulin probe detected a homologous sequence in a wide variety of vertebrate species under stringent conditions. However, no significant hybridization was observed to genomic DNA from invertebrates and microorganisms, even under very low stringency. The sequence and Southern-blot data suggest that the nebulin sequence is highly conserved among vertebrate species.

Amino Acid Sequence↗
PubMed1996

Cloning, expression, and isolation of the mannitol transport protein from the thermophilic bacterium Bacillus stearothermophilus.

A mannitol phosphotransferase system (PTS) was identified in Bacillus stearothermophilus by in vitro complementation with Escherichia coli EI, HPr, and IIA(Mtl). Degenerate primers based on regions of high amino acid similarity in the E. coli and Staphylococcus carnosus EII(Mt1) were used to develop a digoxigenin-labeled probe by PCR. Using this probe, we isolated three overlapping DNA fragments totaling 7.2 kb which contain the genes mtlA, mtlR, mtlF, and mtlD, encoding the mannitol IICB,a regulator, IIA, and a mannitol-1-phosphate dehydrogenase, respectively. The mtl4 gene consists of 1,413 bp coding for a 471-amino-acid protein with a calculated mass of 50.1 kDa. The amino acid sequence shows high similarity with the sequence of IICB(Mtl) of S. carnosus and the IICB part of the IICBA(Mtl)s of E. coli and B. subtilis. The enzyme could be functionally expressed in E. coli by placing it behind the strong tac promoter. The rate of thermal inactivation at 60 degrees C of B. stearothermophilus HCB(Mt1) expressed in E. coli was two times lower than that of E. coli IICB(Mtl). IICB(Mtl) in B. stearothermophilus is maximally active at 85 degrees C and thus very thermostable. The enzyme was purified on Ni-nitrilotriacetic acid resin to greater than 95% purity after six histidines were fused to the C-terminal part of the transporter.

Amino Acid Sequence↗
PubMed1997

The herpes simplex virus type 1 2.0-kilobase latency-associated transcript is a stable intron which branches at a guanosine.

We have used a minigene construct of the herpes simplex virus type 1 (HSV-1) latency-associated transcript (LAT) gene to analyze its transcripts in transient transfection assays. A 2.8-kb fragment of the approximately 8.5-kb LAT gene encompassing the 2.0-kb LAT was cloned into a eukaryotic expression vector downstream of the cytomegalovirus immediate-early gene promoter. Northern hybridization of RNA isolated from transfected COS-1 cells identified three LAT-specific transcripts, 3.4, 2.0, and 1.4 kb in size. Mapping of these transcripts by Northern hybridization indicated that the 1.4- and 2.0-kb RNAs are nonoverlapping, while the 3.4-kb RNA overlaps both smaller RNAs. Reverse transcription-PCR (RT-PCR) and partial sequencing of the 1.4-kb RNA revealed that this RNA is the spliced exons of the 3.4-kb primary transcript. The 2.0-kb LAT appears to be an intron accumulating after splicing of the minor LAT (mLAT) pre-mRNA. The splice donor and acceptor sites for the 2.0-kb LAT identified in transfected and HSV-1-infected cells are identical. Mapping of the branch point of this intron by RT-PCR in transfected and HSV-1-infected cells, as well as in latently infected murine trigemial ganglia, shows that it is a guanosine. This branch site does not bear homology to consensus mammalian branch site sequences. These data provide evidence that the 2.0-kb LAT is an intron of the mLAT pre-mRNA with a unique branch point.

Animals↗