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PubMed1994

An infectious cDNA copy of the genome of a non-cardiovirulent coxsackievirus B3 strain: its complete sequence analysis and comparison to the genomes of cardiovirulent coxsackieviruses.

The genome of the non-cardiovirulent coxsackievirus B3 (CVB3) strain CVB3/0 was cloned and sequenced to aid in the elucidation of the viral genetic basis for the CVB3 cardiovirulent phenotype. Reverse-transcribed sub-genomic complementary DNA (cDNA) fragments were enzymatically amplified using generic oligonucleotide primers and were assembled as a complete infectious genomic copy (pCVB3-0) downstream of the T7 RNA polymerase promoter. Positive-strand viral RNA transcribed from pCVB3-0 using T7 RNA polymerase and transfected into HeLa cells produced infectious virus (CVB3/0c). No differences in phenotype were observed comparing growth of CVB3/0c to the parental CVB3/0 in HeLa single-step growth curves, virus yields, or plaque size. When inoculated into C3H/HeJ mice, CVB3/0c achieved cardiac titers equivalent to the parental CVB3/0 and like the parental virus, demonstrated a non-cardiovirulent phenotype. The nucleotide sequence of the cloned CVB3/0 genome was determined and compared to the genomes of infectious cDNA clones of cardiovirulent CVB3 strains. Two consistent differences among nucleotides in non-translated regions and 8 amino acid differences relative to two well-characterized infectious cDNA copies of genomes from cardiovirulent CVB3 strains were identified.

Animals↗

PubMed1993

Comparative sequence analysis of the coat proteins of biologically distinct citrus tristeza closterovirus isolates.

The genome of citrus tristeza closterovirus (CTV) consists of a 20 kb single-stranded RNA encapsidated in a 2000 nm long, flexuous particle. Double-stranded (replicative form) RNAs purified from CTV-infected tissue were used to prepare complementary DNA libraries that involved initial first-strand cDNA synthesis followed by selective amplification of the coat protein gene. CTV-specific antisera were used to select clones expressing the coat protein. The coat protein genes of seven Florida and four exotic isolates that differ in their biological properties were cloned and sequenced. The gene is 669 base pairs long and encodes a 223 amino acid protein. There was a greater than 80% homology at both nucleotide and amino acid levels among all the isolates examined. However, comparisons showed that each isolate was found to have several unique amino acid residues. Several blocks of amino acid residues were conserved among all the isolates. A cluster dendrogram showed greater similarities among groups of mild and severe Florida isolates that differed significantly from those of the geographically distinct, exotic CTV isolates.

Amino Acid Sequence↗

PubMed1994

Molecular cloning and nucleotide sequence of the coat protein gene of a Cuban isolate of potato leafroll virus and its expression in Escherichia coli.

Total RNA from infected Physalis floridana was isolated to generate complementary DNA corresponding to the coat protein (GP) gene of a Cuban isolate of potato leaf roll virus (PLRV). This cDNA was amplified by the polymerase chain reaction (PCR) and cloned into the bacterial expression vectors pEX(1-3) for fusion protein expression in E. coli. The product was detected by antibodies specific for the PLRV CP. The coding sequence of the CP gene was determined, and the predicted length of the CP was 208 amino acids (23 kD). The nucleotide sequences and deduced amino acid sequences were compared with the other PLRV isolates and found to be 97-99.5% identical at both the nucleotide and amino acid sequence level of other isolates. Comparison of the deduced amino acid sequences of the PLRVcub CP revealed considerable homology to other luteoviruses. We believe that the protocol described could be applicable to other plant viruses of low abundance or of cumbersome isolation, since this method is less time consuming than the traditional methods of cloning coat protein genes of plant viruses with known sequences.

Amino Acid Sequence↗

PubMed2005

Gene structure of the ciliate Sterkiella histriomuscorum based on a combined analysis of DNA and cDNA sequences from 21 macronuclear chromosomes.

Macronuclear deoxyribonucleic acid (DNA) in hypotrichous ciliates consists of a set of linear molecules ranging in size from 0.5 to several tens of kilobases and typically carrying a single gene. Each minichromosome is present at a ploidy of >or=1,000 per macronucleus. These molecules are known as gene-sized molecules. Multigene molecules are also present, but are still poorly described. In analyzing the encystment-excystment cycle of Sterkiella histriomuscorum, we have characterized a set of 21 macronuclear molecules both at the DNA and complementary DNA (cDNA) levels. On a total of 23 validated coding sequences, we mapped the 5' and 3' untranslated regions for a subset of 10 and 18 transcripts, respectively. A combination of DNA and cDNA data allows us to precisely determine several structural features of macronuclear chromosomes, such as the organization of multigene molecules, an intron content higher than expected, and a conserved sequence surrounding the initiation transcription site. It also reveals one coding sequence containing a transcribed 10-bp element that displays the characteristic features of internal eliminated sequences (IES). Its presence in a fraction of the minichromosomes carrying this gene raises the possibility of an incomplete IES excision process during the development of the S. histriomuscorum macronucleus.

3' Untranslated Regions↗

PubMed1995

Monitoring the cDNA synthesis of dengue-2 virus by RT PCR.

Reverse transcriptase polymerase chain reaction (RT PCR) was utilized to observe the complementary DNA (cDNA) synthesis from the 10723 base dengue-2 virus template by in vitro reverse transcription. The PCR product amplified from 5'-end of the genome (PCR primers N1A-E1) indicates the completeness of the cDNA synthesis because the cDNA primer D8B was located at 3'-end and the cDNA synthesized encompassed the entire RNA template. The integrity of the dengue virus RNA was also determined by the cDNA primer extension from 3'-end and the PCR amplification at various regions from 5'-end of the RNA template. In conclusion, RT PCR could be applied to monitor cDNA synthesis by in vitro reverse transcription and measure the integrity of viral RNA template. Moreover, it is demonstrated that at least 10.7 kilobases of the cDNA could be synthesized from dengue-2 virus RNA by in vitro reverse transcription.

Aedes↗

PubMed1995

Cloning and characterization of a putative calcium-transporting ATPase gene from Schistosoma mansoni.

Complementary DNA was isolated, encoding a putative Ca(2+)-transport ATPase (SMA1) of the human parasitic trematode Schistosoma mansoni. The cDNA was isolated by a nested polymerase chain reaction based strategy. The oligonucleotides used were designed on the basis of conserved amino-acid regions found in P-type ATPases. The complete nucleotide sequence was determined. The primary structure and topology of the enzyme were deduced. SMA1 has 1022 amino acids and a predicted molecular mass of 113 kDa. This protein is 67% identical and phylogenetically related to several sarco/endoplasmic reticulum Ca(2+)-ATPases but lacks the phospholamban-binding domain that exists in the SERCA isoforms 1 and 2. The membrane topology predicted for SMA1 is characteristic of the P-type ATPases, showing two major cytoplasmic loops and ten conserved hydrophobic segments. Sequences and residues that are important for the function of the SER Ca(2+)-ATPase, such as the high-affinity Ca(2+)-binding sites, the putative fluorescein isothiocyanate binding site, the 5'-(p-fluorosulfonyl)benzoyladenosine binding site and the aspartyl phosphorylation site, are conserved in SMA1, suggesting that the cloned gene is a Ca(2+)-transport ATPase of the SERCA family. In addition, three PCR products were cloned which share homology with another SER Ca(2+)-ATPase, with the yeast secretory pathway Ca(2+)-ATPase PMR1 and its mammalian homologue, and with the alpha subunit of a Na+,K(+)-ATPase.

Adenosine Triphosphatases↗

PubMed2003

Identification, molecular characterization and expression of the gene encoding the epidermal growth factor receptor orthologue from the fox-tapeworm Echinococcus multilocularis.

Receptor tyrosine kinases (RTKs) are crucially involved in the development of metazoan organisms and possible mediators of cell-cell communication that occurs between eukaryotic parasites and hosts. We have now cloned and characterized the complete complementary DNA (cDNA) molecule encoding a novel receptor tyrosine kinase, EmER, of the human parasite Echinococcus multilocularis. EmER shared significant amino acid sequence homologies with members of the epidermal growth factor (EGF) receptor family of different phylogenetic origin, exhibited a domain structure which is typical for this group of membrane receptors and contained all catalytically important residues at the corresponding positions. Highest homologies were detected between EmER and a previously identified receptor kinase, SER, from the parasitic trematode Schistosoma mansoni. The EmER encoding gene, emer, spans a chromosomal region of 39 kb and is composed of 23 exons. Structural comparisons indicated that emer and the EGF receptor encoding genes from mammals derive from a common ancestor. DNA/DNA hybridization experiments demonstrated that emer is present as a single copy locus in the parasite. Transcriptional analyses on in vitro cultivated parasite larvae revealed that emer is expressed in both the metacestode and the protoscolex stage, although about 10-fold higher transcription levels were detectable for the protoscolex. Northern blot experiments further indicated that emer is expressed as a single 5.2-kb transcript in parasitic larvae during an infection of the intermediate host. These results suggest an involvement of EmER in echinococcal differentiation processes towards the protoscolex stage during natural infections and provide, for the first time, structural information on an epidermal growth factor receptor-like kinase from a cestode.

Amino Acid Sequence↗

PubMed1999

Liver colonization by human colon cancer cells is reduced by antisense inhibition of MUC2 mucin synthesis.

BACKGROUND & AIMS: Alterations in the production of epithelial mucins have been correlated with advanced tumor stage in the colon, but direct evidence for a role of specific mucin genes in liver metastasis is lacking. The current study was designed to establish more directly the role of MUC2 in colon cancer metastasis. METHODS: MUC2 levels were manipulated in highly metastatic human colon cancer cells using eukaryotic expression constructs designed to express a portion of MUC2 complementary DNA in antisense orientation. To assess the effect of MUC2 levels on metastatic potential, liver colonization was assessed in athymic mice after splenic-portal inoculation. RESULTS: Stable integration of the MUC2 antisense construct into metastatic colon cancer cells (LS LiM6) resulted in an 80% reduction in MUC2-specific messenger RNA and a concomitant decrease in MUC2 apomucin protein. This reduction was associated with a 50% reduction in synthesis of mature glucosamine-labeled mucin, almost complete inhibition of secretion of sialyl-LeX and sialyl-Tn antigens, and a 40% decrease in binding of colon cancer cells to E-selectin. Reduction in MUC2 levels was associated with a marked decrease in liver colonization. CONCLUSIONS: This study provides direct evidence that MUC2 plays an important role in colon cancer metastasis.

Animals↗

PubMed2000

Molecular cloning of a ferret angiotensin II AT(1) receptor reveals the importance of position 163 for Losartan binding.

A complementary DNA for the angiotensin II (AngII) type 1 (AT(1)) receptor from Mustela putorius furo (ferret) was isolated from a ferret atria cDNA library. The cDNA encodes a protein (fAT(1)) of 359 amino acids having high homologies (93-99%) to other mammalian AT(1) receptor counterparts. When fAT(1) was expressed in COS-7 cells and photoaffinity labeled with the photoactive analogue (125)I-¿Sar(1), Bpa(8)AngII, a protein of 100 kDa was detected by autoradiography. The formation of this complex was specific since it was abolished in the presence of the AT(1) non-peptidic antagonist L-158,809. Functional analysis indicated that the fAT(1) receptor efficiently coupled to phospholipase C as demonstrated by an increase in inositol phosphate production following stimulation with AngII. Binding studies revealed that the fAT(1) receptor had a high affinity for the peptide antagonist ¿Sar(1), Ile(8)AngII (K(d) of 5. 8+/-1.4 nM) but a low affinity for the AT(1) selective non-peptidic antagonist DuP 753 (K(d) of 91+/-15.6 nM). Interestingly, when we substituted Thr(163) with an Ala residue, which occupies this position in many mammalian AT(1) receptors, we restored the high affinity of this receptor for Dup 753 (11.7+/-5.13 nM). These results suggest that position 163 of the AT(1) receptor does not contribute to the overall binding of peptidic ligands but that certain non-peptidic antagonists such as Dup 753 are clearly dependent on this position for efficient binding.

1-Sarcosine-8-Isoleucine Angiotensin II↗

PubMed2000

Transcriptome analysis of channel catfish (Ictalurus punctatus): genes and expression profile from the brain.

Expressed sequence tag (EST) analysis was conducted using a complementary DNA (cDNA) library made from the brain mRNA of channel catfish (Ictalurus punctatus). As part of our transcriptome analysis in catfish to develop molecular reagents for comparative functional genomics, here we report analysis of 1201 brain cDNA clones. Of the 1201 clones, 595 clones (49.5%) were identified as known genes by BLAST searches and 606 clones (50.5%) as unknown genes. The 595 clones of known gene products represent transcripts of 251 genes. These known genes were categorized into 15 groups according to their biological functions. The largest group of known genes was the genes involved in translational machinery (21.4%) followed by mitochondrial genes (6.2%), structural genes (3.1%), genes homologous to sequences of unknown functions (2.3%), enzymes (2.7%), hormone and regulatory proteins (2.5%), genes involved in immune systems (2.1%), genes involved in sorting, transport, and metal metabolism (1.8%), transcriptional factors and DNA repair proteins (1.6%), proto-oncogenes (1.2%), lipid binding proteins (1.2%), stress-induced genes (0.7%), genes homologous to human genes involved in mental diseases (0.6%), and development or differentiation-related genes (0.3%). The number of genes represented by the 606 clones of unknown genes is not known at present, but the high percentage of clones showing no homology to any known genes in the GenBank databases may indicate that a great number of novel genes exist in teleost brain.

Animals↗

PubMed2001

Neutral liposome-mediated delivery process of fluorescein-modified oligonucleotides in cultured human keratinocytes.

We propose a model of the intracellular delivery process in which fluorescein-labeled natural oligonucleotides (F-DNA) are transferred into the nuclei of cultured human keratinocytes. By encapsulation in neutral multilamellar lecithin liposomes, the F-DNA appeared to be protected against intracellular interactions with cellular materials and nuclease attacks in the cytoplasm during the process. The intracellular behavior of F-DNA and fluorescent phospholipid-labeled liposomes was observed by means of fluorescence analysis. Results showed that: F-DNA encapsulated in neutral multilamellar liposomes reached the cellular nuclei more efficiently than either free F-DNA, or F-DNA in unilamellar liposomes; the liposomal membranes appeared to be left in the cytoplasm. The reaction of F-DNA with complementary DNA was suggested by a rapid quenching of the fluorescence in the nucleus. In addition, the fluorescence decrease was evidently suppressed in the cytoplasm, indicating a protective effect of the neutral multilamellar liposomes against the interaction of F-DNA with cytoplasmic materials. The application of these findings to 'photo'-antisense studies has been discussed, where suppression of a gene expression is attempted by using oligonucleotide-attached fluorescein with the aid of a photo-induced covalent binding property.

Binding Sites↗

PubMed1978

Measurement of the complexity and diversity of poly(adenylic acid) containing messenger RNA from rat liver.

The complexity of rat liver poly (A)+ messenger RNA (mRNA) has been measured by analysis of the kinetics of hydridization with both complementary DNA (cDNA) and single copy DNA. The complementary DNA-poly(A)+ mRNA hybridization reaction demonstrates the existence of three abundance classes representing 18, 37, and 45% of the cDNA and 4, 290, and 24 000 different 1800-nucleotide sequences respectively. The poly(A)+ mRNA driven single copy DNA hybridization reaction reveals a single major transition accounting for 1.9% of the haploid rat genome. The kinetics of the poly(A)+ mRNA driven single copy DNA reaction suggest that approximately 45% of the mass of the mRNA population contains over 95% of the complexity. Although higher than previous estimates, the base sequence complexities of rat liver poly(A)+ mRNA measured in these two ways are in good agreement, suggesting that the technique of poly(A)+ mRNA-cDNA hybridization may be used in approximating the complexity as well as abundance of a messenger RNA population. DNA-driven cDNA reactions reveal that about 10% of rat liver poly(A)+ mRNA is transcribed from repetitive sequences in the rat genome.

Animals↗

PubMed2001

Transcriptional activation of a maize calcium-dependent protein kinase gene in response to fungal elicitors and infection.

Plants respond to pathogen infection with the activation of the expression of pathogenesis-related genes, a response that involves Ca2+-regulated protein phosphorylation processes. We report here the isolation of a full-length complementary DNA encoding a calcium-dependent protein kinase (CPK) gene from maize. CPK genes occur in maize as members of a multigene family, but only one specific CPK gene, the ZmCPK10 gene here described, is transcriptionally activated in response to both fungal infection and treatment with fungal elicitors. Activation of the ZmCPK10 gene is extremely rapid. ZmCPK10 transcripts could be detected 5 min after elicitation and reached maximum levels at 30 min after treatment. Afterwards, there was a decline in the level of ZmCPK10 transcripts followed by a basal level of accumulation which is maintained over the time period of elicitor treatment. The activation of this kinase is accompanied by an increase in the level of PRms mRNA, the PRms being a pathogenesis-related protein from maize whose expression is induced in maize tissues in response to fungal infection and treatment with fungal elicitors. In situ mRNA hybridization analysis revealed a remarkable cell-type specific pattern of expression of ZmCPK10 during growth and development of the elicitor-treated or fungus-infected seedling. Moreover, the ZmCPK10 gene is expressed only in those specific cell types in which the PRms gene is also expressed. The involvement of ZmCPK10 in the elicitor-induced signal transduction pathway leading to the activation of PRms gene expression is discussed.

Amino Acid Sequence↗

PubMed1997

Analysis of the fusion protein gene of the porcine rubulavirus LPMV: comparative analysis of paramyxovirus F proteins.

Complementary DNA clones representing the fusion (F) protein gene of the porcine rubulavirus LPMV were isolated and sequenced. The F gene was found to be 1,845 nucleotides long containing one long open reading frame capable of encoding a protein of 541 amino acids. The cleavage motif for F0 into F1 and F2 is His-Arg-Lys-Lys-Arg. A sequence comparison and a phylogenetic analysis was performed in order to identify possible functional domains of paramyxovirus fusion proteins and also to classify the porcine rubulavirus. The F gene of LPMV is most closely related to the human mumps virus and simian virus type 5 F genes, and is therefore classified into the rubulavirus genus. A coding region for a small hydrophobic protein was however not found between the F and hemagglutinin-neuraminidase (HN) genes as previously found in both SV5 and mumps.

Amino Acid Sequence↗

PubMed2001

Delineation of prognostic biomarkers in prostate cancer.

Prostate cancer is the most frequently diagnosed cancer in American men. Screening for prostate-specific antigen (PSA) has led to earlier detection of prostate cancer, but elevated serum PSA levels may be present in non-malignant conditions such as benign prostatic hyperlasia (BPH). Characterization of gene-expression profiles that molecularly distinguish prostatic neoplasms may identify genes involved in prostate carcinogenesis, elucidate clinical biomarkers, and lead to an improved classification of prostate cancer. Using microarrays of complementary DNA, we examined gene-expression profiles of more than 50 normal and neoplastic prostate specimens and three common prostate-cancer cell lines. Signature expression profiles of normal adjacent prostate (NAP), BPH, localized prostate cancer, and metastatic, hormone-refractory prostate cancer were determined. Here we establish many associations between genes and prostate cancer. We assessed two of these genes-hepsin, a transmembrane serine protease, and pim-1, a serine/threonine kinase-at the protein level using tissue microarrays consisting of over 700 clinically stratified prostate-cancer specimens. Expression of hepsin and pim-1 proteins was significantly correlated with measures of clinical outcome. Thus, the integration of cDNA microarray, high-density tissue microarray, and linked clinical and pathology data is a powerful approach to molecular profiling of human cancer.

Biomarkers, Tumor↗

PubMed1995

Presence of a heterozygous substitution and its relationship to DT-diaphorase activity.

A point mutation in the mRNA of NADP(H): quinone oxidoreductase 1 (NQO1, DT-diaphorase) is believed to be responsible for reduced enzyme activity in the adenocarcinoma BE cell line. The present study examined nine cultured human non-cancerous fibroblast cell strains, five of which were from members of a single cancer-prone family, which demonstrated widely varying activity levels of DT-diaphorase (41 - 3462 nmol min-1 mg-1 protein), to determine if genetic alteration of the NQO1 or NOQ2 gene was involved in determining enzyme activity. All cell strains expressed NQO1 and NQO2 mRNA as measured by a quantitative polymerase chain reaction amplification technique. No relationship was found between the level of mRNA expressed and the enzyme activity in the cells. Sequencing of the entire complementary DNA from the cell strains revealed only a single base substitution at nucleotide 609 in one allele encoding NQO1 in every cell strain from members of the cancer-prone family, except for one cell strain which expressed only the T at nucleotide 609 in both alleles. Subsequent examination of genomic DNA from 44 individuals revealed that this base substitution is present in approximately 50% of the population. The presence of the T at nucleotide 609 in the NQO1 locus does not appear to be directly causal for altered DT-diaphorase activity.

Adenocarcinoma↗

PubMed2006

The inner-nuclear-envelope protein emerin regulates HIV-1 infectivity.

Primate lentiviruses such as human immunodeficiency type 1 (HIV-1) have the capacity to infect non-dividing cells such as tissue macrophages. In the process, viral complementary DNA traverses the nuclear envelope to integrate within chromatin. Given the intimate association between chromatin and the nuclear envelope, we examined whether HIV-1 appropriates nuclear envelope components during infection. Here we show that emerin, an integral inner-nuclear-envelope protein, is necessary for HIV-1 infection. Infection of primary macrophages lacking emerin was abortive in that viral cDNA localized to the nucleus but integration into chromatin was inefficient, and conversion of viral cDNA to non-functional episomal cDNA increased. HIV-1 cDNA associated with emerin in vivo, and the interaction of viral cDNA with chromatin was dependent on emerin. Barrier-to-autointegration factor (BAF), the LEM (LAP, emerin, MAN) binding partner of emerin, was required for the association of viral cDNA with emerin and for the ability of emerin to support virus infection. Therefore emerin, which bridges the interface between the inner nuclear envelope and chromatin, may be necessary for chromatin engagement by viral cDNA before integration.

Cells, Cultured↗

PubMed1995

Mutation of the androgen-receptor gene in metastatic androgen-independent prostate cancer.

BACKGROUND: Metastatic prostate cancer is a leading cause of cancer-related death in men. The rate of response to androgen ablation is high, but most patients relapse as a result of the outgrowth of androgen-independent tumor cells. The androgen receptor, which binds testosterone and stimulates the transcription of androgen-responsive genes, regulates the growth of prostate cells. We analyzed the androgen-receptor genes from samples of metastatic androgen-independent prostate cancers to determine whether mutations in the gene have a role in androgen independence. METHODS: Complementary DNA was synthesized from metastatic prostate cancers in 10 patients with androgen-independent prostate cancer, and the expression of the androgen-receptor gene was estimated by amplification with the polymerase chain reaction. Exons B through H of the gene were cloned, and mutations were identified by DNA sequencing. The functional effects of the mutations were assessed in cells transfected with mutant genes. RESULTS: All androgen-independent tumors expressed high levels of androgen-receptor gene transcripts, relative to the levels expressed by an androgen-independent prostate-cancer cell line (LNCaP). Point mutations in the androgen-receptor gene were identified in metastatic cells from 5 of the 10 patients examined. One mutation was in the same codon as the mutation found previously in the androgen-independent prostate-cancer cell line. The mutations were not detected in the primary tumors from of the two patients. Functional studies of two of the mutant androgen receptors demonstrated that they could be activated by progesterone and estrogen. CONCLUSIONS: Most metastatic androgen-independent prostate cancers express high levels of androgen-receptor gene transcripts. Mutations in androgen-receptor genes are not uncommon and may provide a selective growth advantage after androgen ablation.

Base Sequence↗