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At least 19 recordsLinked to original sources
PubMed1994

Construction and characterization of infectious human T-cell leukemia virus type 1 molecular clones.

Molecular clones of human T-cell leukemia virus type 1 (HTLV-1) have been constructed and stably propagated in plasmids in Escherichia coli. Expression of Tax could be demonstrated from these clones in fibroblast and epithelial cell lines. HOS cells stably transfected with HTLV-1 clone ACH produced all three classes of viral transcripts and Gag proteins. Virus-like particles were also produced from ACH transfected HOS cells as demonstrated by sucrose gradient and electron microscopic analyses. Transfection of peripheral blood mononuclear cells with ACH resulted in production of infectious virus particles which induced lymphocyte proliferation. This study describes useful reagents for further examination of the biological properties of HTLV-1.

Base Sequence↗
PubMed1995

Unique reactive site domains of neuroendocrine isoforms of alpha 1-antichymotrypsin from bovine adrenal medulla and pituitary revealed by molecular cloning.

Molecular cloning of bovine adrenal medulla (AM) and pituitary (Pit) alpha 1-antichymotrypsin cDNAs indicated novel isoforms of ACT. The deduced primary sequences indicated that the AM ACT and Pit ACT possess COOH-terminal reactive-site domains that are characteristic of serpins (serine protease inhibitors). Of high interest was the finding of unique reactive sites within AM ACT and Pit ACT which are predicted to possess Arg as P1 residue. Arginine as P1 residue parallels the cleavage specificity of neuroendocrine prohormone processing enzymes cleaving at basic residues. Furthermore, RT-PCR indicated tissue-specific expression of AM and Pit ACT mRNAs. The AM and Pit isoforms of ACT may regulate novel target proteases involved in neuroendocrine function.

Adrenal Medulla↗
PubMed1992

Molecular clones from a non-acutely pathogenic derivative of SIVsmmPBj14: characterization and comparison to acutely pathogenic clones.

Molecularly cloned simian immunodeficiency viruses capable of inducing acute, fatal disease in pig-tailed macaques had been derived previously from a biological clone (bcl-3) of the PBj14 isolate of SIV from sooty mangabey monkeys (SIVsmmPBj14). The present study was undertaken in order to characterize virus from a second biological clone of SIVsmmPBj14, bcl-1, which fails to induce acute or fatal disease. Polymerase chain reaction was used to amplify 5' and 3' viral genome halves. The DNA sequence of two 3' halves was determined, and an infectious recombinant generated using a bcl-3-derived 5' half and a bcl-1-derived 3' half. Overall, bcl-1- and bcl-3-derived viruses displayed close homology, differing by a total of 2% at the DNA level and 1-6% at the amino acid level within the 8 open reading frames examined. In contrast to the bcl-3-derived viruses, the bcl-1-derived viruses encode a truncated transmembrane envelope glycoprotein. Another consistent difference was the presence of a 22 bp duplication in the U3 portion of the long terminal repeat (LTR) of bcl-3-derived viruses that includes the NF-kappa B transcriptional enhancer binding site. To assess the importance of this duplication, virus chimeras were generated which removed the duplication from the 3'-LTR or from both LTRs of a bcl-3 clone. The former virus was unstable, reacquiring the duplication through recombination with the 5' LTR. No consistent difference were observed, however, between viruses with or without the duplication in the in vitro studies conducted to date.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed2000

A strategy for cloning infectious molecular clones of retroviruses from serum or plasma.

To enable biological characterisation of lentiviral variants which emerge during infection and development of AIDS, a method was developed to construct molecular clones from circulating simian immunodeficiency virus (SIV) particles present in as little as 20 microl of serum from infected rhesus monkeys. This technique uses a long distance RT-PCR method optimised for the amplification of partly overlapping 5-kb SIV (half genome) amplimers. Ligation of the genome halves resulted in the construction of full-length clones which, after transfection, were able to replicate well in rhesus peripheral blood mononuclear cells (PBMCs) and in various human T-cell lines inducing syncytia. In addition to the study of molecular cloned virus quasispecies emerging in circulation as a result of immune escape, this method may also be applied to obtain entire genes or full-length molecular clones. These clones may be present in other extracellular body fluids such as urine, saliva, tears, lymph, and bronchial or cerebral spinal fluid. Genes amplified in this way can be inserted quickly in new recombinant expression vectors and may then be applied for DNA vaccination approaches.

AIDS Vaccines↗
PubMed1986

Molecular cloning of the Mason-Pfizer monkey virus genome: biological characterization of genome length clones and molecular comparisons to other retroviruses.

The molecular cloning of the DNA provirus of Mason-Pfizer monkey virus (M-PMV) is described. Fourteen independent clones of integrated M-PMV proviruses were isolated from a human embryo kidney cell line that had been previously derived from a single cell clone infected with M-PMV. Characterization of these clones for size of insert, restriction pattern of flanking DNA, and presence of repetitive DNA in the flanking sequences revealed that 10 of the isolates were identical while the four remaining clones were unique. Three independent clones of unintegrated M-PMV proviruses containing a single copy of the long terminal repeat (LTR) were cloned from acutely infected human embryo kidney cells, Transfection assays revealed that 13 of 14 integrated proviruses and 2 of 3 unintegrated proviruses were capable of producing infectious virus. One of the integrated provirus clones (clone 6A) produced consistently higher titers of virus than all of the other clones in all assays used and in two different cell lines, indicating that it contained a mutation that enhances virus replication. The virus recovered after transfection was shown to be capable of inducing cell fusion in nontransformed cell lines, confirming that this property is associated with M-PMV. One of the clones was hybridized under conditions of varying stringency, to molecular clones of type B, C, and D retroviruses. These studies revealed M-PMV to be most closely related to squirrel monkey retrovirus (D-type virus) and more distantly related to mouse mammary tumor virus (B-type virus). Hybridization was also detected with clones from the pol gene region of a family of human endogenous sequences. No homology was detected with Rous sarcoma virus or most mammalian C-type viruses tested. The exceptions were baboon endogenous virus and RD114 in which previously identified homology in the env gene was confirmed. These results suggest that the type D and type B viruses can be linked together in a group of viruses of similar ancestral origin analogous to that recently proposed for the human T-cell leukemia viruses and bovine leukemia virus.

Animals↗
PubMed2003

Analysis of zinc-fingers and homeoboxes (ZHX)-1-interacting proteins: molecular cloning and characterization of a member of the ZHX family, ZHX3.

Human zinc-fingers and homeoboxes (ZHX) 1, a transcriptional repressor, was originally cloned as an interacting protein with the activation domain of the A subunit of nuclear factor-Y (NF-YA). As the first step in investigating the mechanism by which ZHX1 acts as a transcriptional repressor, we conducted a search of ZHX1-interacting proteins using a yeast two-hybrid system. Nuclear proteins such as ZHX1, transcriptional co-factors and DNA-binding proteins, zyxin, androgen-induced aldose reductase and eleven-nineteen lysine-rich leukaemia gene, as well as some unknown proteins, were cloned. Molecular cloning and determination of the nucleotide sequence of the full-length cDNA encoding a novel protein revealed that it consists of 956 amino acid residues and contains two zinc-finger (Znf) motifs and five homeodomains (HDs) as well as ZHX1. We concluded that the protein forms the ZHX family with ZHX1 and denoted it ZHX3. ZHX3 not only dimerizes with both ZHX1 and ZHX3, but also interacts with the activation domain of the NF-YA. Further analysis revealed that ZHX3 is a ubiquitous transcriptional repressor that is localized in nuclei and functions as a dimer. Lastly, the dimerization domain, the interaction domain with NF-YA, and the repressor domain are mapped to a region including the HD1 region, and two nuclear localization signals are mapped to the N-terminal through Znf1 and the HD2 region, respectively.

Amino Acid Sequence↗
PubMed1984

Molecular cloning of Rauscher spleen focus-forming virus and biological properties of the cloned virus.

Rauscher virus (RV) induces acute erythroleukaemia and a myeloproliferative disease in adult mice. It consists of a replication-competent murine leukaemia virus (R-MuLV) which acts as a helper virus and a defective transforming component which causes spleen focus formation, Rauscher spleen focus-forming virus (R-SFFV). The integrated proviral DNA of R-SFFV was cloned molecularly. The cloned R-SFFV was compared to that of other viral components which are associated with RV-induced disease and also the cloned Friend SFFV (F-SFFV) and the myeloproliferative sarcoma virus (MPSV), both of which expand the erythroid (F-SFFV, MPSV) and myeloid (MPSV) compartment on infection of adult mice. The genome of R-SFFV differs, if analysed by restriction enzymes, from R-MuLV in the 3' end of the genome between the env gene and the long terminal repeat. The difference is most likely an alteration in the 3' part of the gp70-coding region of the env gene. Comparison with Rauscher mink cell focus-inducing virus (R-MCF) suggests that R-SFFV is derived from R-MCF by substitution of the 3' half of the env gene with a sequence of unknown origin. The molecularly cloned R-SFFV pseudotyped with Friend MuLV induces an increase in late erythroid precursor cells which still require erythropoietin for maturation. Host range studies of the molecularly cloned R-SFFV prove that the Fv-2r locus is required but not sufficient to restrict RV-induced haemopoiesis in adult mice, thus suggesting that R-SFFV has a different target cell range than F-SFFV and is similar to MPSV.

Animals↗
PubMed1999

Isolation, characterization, molecular cloning and molecular modelling of two lectins of different specificities from bluebell (Scilla campanulata) bulbs.

Two lectins have been isolated from bluebell (Scilla campanulata) bulbs. From their isolation by affinity chromatography, they are characterized as a mannose-binding lectin (SCAman) and a fetuin-binding lectin (SCAfet). SCAman preferentially binds oligosaccharides with alpha(1,3)- and alpha(1,6)-linked mannopyranosides. It is a tetramer of four identical protomers of approx. 13 kDa containing 119 amino acid residues; it is not glycosylated. The fetuin-binding lectin (SCAfet), which is not inhibited by any simple sugars, is also unglycosylated. It is a tetramer of four identical subunits of approx. 28 kDa containing 244 residues. Each 28 kDa subunit is composed of two 14 kDa domains. Both lectins have been cloned from a cDNA library and sequenced. X-ray crystallographic analysis and molecular modelling studies have demonstrated close relationships in sequence and structure between these lectins and other monocot mannose-binding lectins. A refined model of the molecular evolution of the monocot mannose-binding lectins is proposed.

Amino Acid Sequence↗
PubMed1990

[Molecular cloning and molecular biological studies of human thymidylate synthase gene].

Thymidylate synthase (TS) provides the only de novo source of thymidylate for deoxyribonucleic acid (DNA) synthesis and is a key target of cancer chemotherapeutic agents. Expression of its activity is strongly dependent on the cell cycle. Therefore, it is important for understanding the abnormal growth of cancer cells as well as for cancer chemotherapy to elucidate the structure of a TS gene and the regulatory mechanism of expression of its gene at the molecular level. From this point of view, genomic DNA segments partially coding for human TS were cloned from the mouse cell transformant obtained by gene transfer. By using them as a probe, functional cDNA and genomic DNA (approx. 18 kb) clones for human TS were isolated, and the entire nucleotide sequence of these cloned DNAs including all the introns determined. On the basis of the sequence data, we revealed an amino acid sequence of human TS, and organization and several structural features of its gene were revealed. Then, multiple transcription initiation sites and transcriptional regulatory elements of the TS gene were identified. Furthermore, the importance of the post-transcriptional regulation in the cell-cycle dependent expression of the gene was shown. The results obtained so far have opened the way to elucidate the molecular mechanism regulating the TS gene expression entirely.

Amino Acid Sequence↗
PubMed2001

Rapid extraction and purification of environmental DNA for molecular cloning applications and molecular diversity studies.

A rapid method for the extraction and purification of DNA from environmental samples for molecular cloning applications was developed. The indigenous cells from plant debris, organic materials, sediments, and soils were lysed directly by using DAS-IZ solution and the nucleic acids were precipitated with isopropanol. A simple purification step using DAS-IIZ solution without binding matrix produced highly pure, colorless and undegraded DNA with molecular weight of more than 20 kb. The superiority of this method was tested for wide applications in molecular cloning, i.e., construction of genomic library by using Lambda DASHII Vector and GigapackIII XL, plasmid library, cloning of gene encoding protease, and molecular microbial diversity analysis. An additional advantage of this method is that only 0.1 g of sample is required, if analysis of many samples in short time should be done. To extract large amounts of environmental DNA for molecular cloning lasts only 30 min and to purify it less than 1 h.

Cloning, Molecular↗
PubMed2001

Thymic lesions in cats infected with a pathogenic molecular clone or an ORF-A/2-deficient molecular clone of feline immunodeficiency virus.

Previous studies using feline immunodeficiency virus (FIV) molecular clones lacking the putative transactivator gene (ORF-A/2) failed to address the issue of thymus pathogenesis or investigate the levels of viral replication in separate lymphoid compartments (Y. Inoshima, et al., J. Virol. 70:8518-8526, 1996; E. E. Sparger, et al., Virology 205:546-553, 1994). Using a highly pathogenic molecular clone of FIV, JSY3, and an ORF-A/2-deficient mutant, JSY3DeltaORF-A/2, we compared viral replication and the extent of thymic dysfunction as measured by the formation of lymphoid follicles and alteration of the thymocyte subsets. Viral replication was reduced in JSY3DeltaORF-A/2-infected cats as measured by lymphocyte coculture, immunohistochemistry, and quantitative PCR. Cell-associated viral load measured by lymphocyte coculture varied in a tissue-dependent manner with replication highest in lymphocytes isolated from the thymus, lower in those from the peripheral blood, and lowest in those from lymph node. Thymic proviral load and the number of viral p24 Gag-positive cells within the thymus detected by immunohistochemistry were also reduced. In addition, the onset of a reduced peripheral blood CD4/CD8 ratio was delayed in JSY3DeltaORF-A/2-infected cats. The formation and extent of thymic lymphoid follicular hyperplasia were similar in JSY3 and JSY3DeltaORF-A/2-infected cats as measured by anticytokeratin immunohistochemistry and flow cytometry for percent pan T-negative, immunoglobulin G-positive cells within the thymus. In contrast, comparison of thymocyte subpopulations demonstrated a reduced expansion of single-positive CD4(-) CD8(+) thymocytes in JSY3DeltaORF-A/2-infected cats. Level of viral replication, therefore, may not correlate with the formation of thymic lymphoid follicles but may correlate with the expansion of the single-positive CD4(-) CD8(+) thymocyte subpopulation.

Animals↗
PubMed1994

Bovine connexin44, a lens gap junction protein: molecular cloning, immunologic characterization, and functional expression.

PURPOSE: To identify, clone molecularly, characterize immunochemically, and express functionally a bovine lens gap junction protein (connexin). METHODS: The methods used were polymerase chain reaction, genomic cloning, RNA and DNA blotting, bacterial expression of a fusion protein, immunoblotting, alkaline phosphatase treatment, Xenopus oocyte expression, and voltage clamp technique. RESULTS: A bovine genomic clone encoding a polypeptide of 44,424 d, termed connexin44 (Cx44), was isolated. Cx44 was most closely related to the lens connexins rat Cx46 and chicken Cx56. The Cx44 DNA hybridized to a 2.5 kb mRNA detected only in lens RNA. The carboxyl terminal 161 amino acids from Cx44 were expressed in bacteria fused to maltose binding protein (MBP). The Cx44/MBP fusion protein reacted in immunoblots with anti-rat Cx46(411 to 416) antibodies and with the monoclonal antibody 5H1, but not with a monoclonal antibody to MP70 nor with antibodies to other connexins. Cx44 translated in vitro from the cloned DNA showed a single band with an apparent electrophoretic mobility of approximately 50 kd on polyacrylamide gels containing sodium dodecyl sulfate. Multiple bands of 53 to 57 kd were detected by immunoblotting in homogenates of bovine lens; these bands were reduced to a broad band of approximately 50 kd by alkaline phosphatase treatment, suggesting that they represented phosphorylated forms of Cx44. Cx44 RNA injected in single oocytes induced a large and characteristic time- and voltage-dependent current. Overexpression of Cx44 produced depolarization and cell lysis. Junctional currents that could be regulated by transjunctional voltage were induced between paired oocytes injected with Cx44 RNA. Observations in paired oocytes suggested the assembly of hemichannels into junctional channels. CONCLUSIONS: Cx44 is a phosphoprotein component of bovine lens fiber gap junctions. Although it has a relatively distinct sequence, it shares sequence similarity, immunologic cross-reactivity, and electrophysiological properties with rat Cx46. These data suggest that Cx44 is the protein previously identified in several immunohistochemical studies of bovine lens gap junctions that used anti-rat Cx46 antibodies. They also suggest that the formation of intercellular channels by pairing of hemichannels might prevent the cell lysis induced by the opening of unpaired hemichannels.

Amino Acid Sequence↗
PubMed1990

Molecular cloning and DNA sequence analysis of Escherichia coli priA, the gene encoding the primosomal protein replication factor Y.

Escherichia coli replication factor Y (protein n') functions in the assembly of a mobile multiprotein replication-priming complex called the primosome. Although the role of factor Y in primosome assembly during replication in vitro of bacteriophage phi X174 and plasmid pBR322 DNA is clear, its role in E. coli chromosomal replication is not. To address this issue, the gene for factor Y has been cloned molecularly and its DNA sequence has been determined. The cloned fragment of DNA contained an open reading frame capable of encoding a polypeptide of 81.7 kDa. This open reading frame contains amino acid sequences identical to 13 N-terminal amino acids of purified factor Y, as well as to a 10-amino acid internal sequence (from a cyanogen bromide fragment) as determined by gas-phase microsequencing. Expression of the polypeptide encoded by this open reading frame using a bacteriophage T7 transient expression system resulted in the accumulation of a polypeptide with an apparent molecular mass of 78 kDa that comigrated with bona fide factor Y during SDS/polyacrylamide gel electrophoresis. Soluble extracts made from cells overexpressing the product of the putative factor Y open reading frame showed a 2000-fold increase in factor Y activity during bacteriophage phi X174 complementary-strand DNA synthesis in vitro when compared to control extracts. The gene encoding factor Y, which maps to 88.5 min on the E. coli chromosome, has been designated primosome A (priA).

Amino Acid Sequence↗
PubMed1988

Characterization of infectious molecular clones of simian immunodeficiency virus (SIVmac) and human immunodeficiency virus type 2: persistent infection of rhesus monkeys with molecularly cloned SIVmac.

Infection of macaque monkeys with simian immunodeficiency virus (SIV) is probably the best animal model currently available for studying acquired immunodeficiency syndrome. In this report, we describe three infectious molecular clones of SIVmac and one of human immunodeficiency virus type 2 (HIV-2) and their use in the study of cell and species specificity, animal infection, and the relationship of gene sequence to function. Replication of the cloned viruses in different cell lines varied dramatically. Some human CD4+ cell lines (HUT 78 and MT-4) supported the replication of SIVmac and HIV-2, while others (CEM and Jurkat-T) supported the replication of HIV-2 but not SIVmac. Growth of cloned virus in macaque lymphocytes in vitro was predictive of macaque infection in vivo. Macaque lymphocytes supported the replication of SIVmac239 and SIVmac251 but not SIVmac142 or HIV-2ROD. Using virus recovery and antibody response as criteria for infection, macaques that received cloned SIVmac251 and SIVmac239 became infected, while macaques receiving cloned SIVmac142 and HIV-2ROD did not become infected. Nucleotide sequences from the envelope region of all four cloned viruses demonstrated that there is considerable flexibility in the location of the translational termination (stop) signal. These infectious molecular clones will be very useful for future studies directed at the molecular basis for persistence, pathogenicity, tropism, and cell and species specificity.

Amino Acid Sequence↗
PubMed1992

The thyrotropin receptor 25 years after its discovery: new insight after its molecular cloning.

The molecular cloning and functional expression of the TSH receptor has led to rapid advances in understanding the structure and function of the molecule. Knowledge of its genomic structure provides information on the evolutionary origin of the TSH receptor as well as on the functional organization of its extracellular domain, which is responsible for ligand binding. A beginning has been made in defining the discontinuous contact points for TSH in this extracellular region, but determination of all of the amino acids involved will be difficult. The binding sites of TSH receptor autoantibodies do not appear to be identical to the TSH binding site. Two of the six potential glycosylation sites in the extracellular domain are important in the expression of a functional receptor. Disulfide bonding contributes toward maintenance of the three-dimensional structure of the receptor. Recent evidence suggests that the TSH receptor exists as a single polypeptide chain without subunits. Significant progress has been made in understanding the intracellular regions of the TSH receptor that are involved in signal transduction. Although still in the distant future, we are closer to the goal of understanding precisely how TSH interacts with and activates its receptor. More importantly from the clinical perspective, we are closer to defining the B cell, and ultimately T cell, epitopes on the TSH receptor that are recognized by the immune system. This information may ultimately facilitate the development of immunological approaches to treating Graves' disease, which will be an improvement over thyroid gland destruction and consequent hypothyroidism, the most common form of therapy at the present time.

Amino Acid Sequence↗
PubMed1992

Molecular cloning and characterization of human endothelial nitric oxide synthase.

The constitutive calcium/calmodulin-dependent nitric oxide (NO) synthase expressed in vascular endothelium shares common biochemical and pharmacologic properties with neuronal NO synthase. However, recent cloning and molecular characterization of NO synthase from bovine endothelial cells indicated the existence of a family of constitutive NO synthases. Accordingly, we undertook molecular cloning and sequence analysis of human endothelial NO synthase. Complementary DNA clones predict a protein of 1,203 amino acids sharing 94% identity with the bovine endothelial protein, but only 60% identity with the rat brain NO synthase isoform. Northern blot analysis with an endothelial-derived cDNA identified a 4.6-4.8 kb mRNA transcript in HUVEC and in situ hybridization localized transcripts to vascular endothelium but not neuronal tissue.

Amino Acid Oxidoreductases↗
PubMed1992

Molecular cloning of a new variant of human papillomavirus type 6 isolated from a Chinese woman and expression of its L1 gene in E. coli--molecular cloning & expression of HPV6 gene.

A human papillomavirus genome DNA of 7.9 kb from a Chinese woman with genital condyloma acuminata was cloned in BamHI site of pAT153. According to the results obtained from Southern blotting, restriction mapping as well as partial DNA sequencing, the isolated genome (HPV6BV) had obvious variance and was referred to as a new variant of HPV6 found in China the first time. HPV6BV L1 gene was successfully expressed in E. coli as a fusion protein with pUR288. The beta-galactosidase/L1 fusion protein reacted with both beta-galactosidase antiserum and HPV antibody using Western blot technique. The E. coli-produced fusion protein, possessing HPV antigenicity, may provide a reagent for clinical diagnosis and epidemiological survey.

Cloning, Molecular↗