Biomedical subjects

N Tanese

Publications and source records attributed to N Tanese.

30 records · Page 2Linked to original sources

PubMed1990

Functional domains and upstream activation properties of cloned human TATA binding protein.

The TATA binding protein, TFIID, plays a central role in the initiation of eukaryotic mRNA synthesis. Here, we present a human cDNA clone for this factor. Comparison of its predicted protein sequence with those from Drosophila and yeast reveals a highly conserved carboxyl-terminal 180 amino acids. By contrast, the amino-terminal region of TFIID has diverged in both sequence and length. A striking feature of the human protein is a stretch of 38 glutamine residues in the NH2-terminal region. Expression of human TFIID in both Escherichia coli and HeLa cells produces a protein that binds specifically to a TATA box and promotes basal transcription; the conserved COOH-terminal portion of the protein is sufficient for both of these activities. Recombinant TFIID forms a stable complex on a TATA box either alone or in combination with either of the general transcription factors, TFIIA or TFIIB. Full-length recombinant TFIID is able to support Sp1 activated transcription in a TFIID-depleted nuclear extract, while a deletion of the NH2-terminal half of the protein is not. These results indicate the importance of the NH2-terminal region for upstream activation functions and suggest that additional factors (co-activators) are required for mediating interactions with specific regulators.

Amino Acid Sequence↗

PubMed1990

Analysis of mutations in the integration function of Moloney murine leukemia virus: effects on DNA binding and cutting.

The 3' terminus of the pol gene of Moloney murine leukemia virus encodes the integration (IN) protein, required for the establishment of the integrated provirus. A series of six linker insertion mutations and two single-base substitutions were generated within the region encoding the IN protein. Mutations were initially generated within an Escherichia coli plasmid expressing the IN protein, and the resulting variants were assayed for DNA-binding activity. Mutations which altered conserved cysteine residues within a potential DNA finger-binding motif resulted in lower or variable DNA binding, which appeared to be the result of variable protein folding. Upon renaturation, these proteins were able to nonspecifically bind DNA in a manner similar to that of the other mutant IN proteins and the parent. When reconstructed back into full-length virus, seven of the eight mutations were lethal. All mutants produced a stable IN protein in virions and mediated normal conversion of the retroviral RNA to its three DNA forms. Fine-structure analysis of the linear double-stranded viral DNA indicated that all seven lethal alterations within the IN protein blocked the formation of the 3' recessed termini that normally precedes integration.

Amino Acid Sequence↗

PubMed1989

An insertion mutation in the pol gene of Moloney murine leukemia virus results in temperature-sensitive pol maturation and viral replication.

An insertion mutation in the pol gene of Moloney murine leukemia virus (M-MuLV) was found to render the virus temperature-sensitive for replication. A provirus containing a 12-bp insertion at the boundary between the reverse transcriptase (RT) and integrase (IN) domains induced the formation of mutant virions containing a partially processed RT-IN fusion protein. Some proteolytic processing to form mature RT and IN was observed at 32 degrees, but only aberrantly processed proteins were detected at 39 degrees. The uncleaved precursor was found to exhibit DNA polymerase activity, even though it could not support replication of the virus in vivo at 39 degrees.

Animals↗

PubMed1988

Gene product of Moloney murine leukemia virus required for proviral integration is a DNA-binding protein.

The 3'-terminal portion of the retroviral pol gene encodes a function required for the formation of the integrated provirus soon after infection of sensitive cells. To permit the isolation of large quantities of the gene product, we expressed various portions of the pol gene of Moloney murine leukemia virus (M-MuLV) as trpE fusion proteins in Escherichia coli. The proteins were found to exhibit strong DNA-binding activity after extraction and renaturation by two different procedures. In the first method, proteins separated by polyacrylamide gel electrophoresis were blotted to nitrocellulose and assayed when bound to the support. The second procedure involved the isolation of proteins in an insoluble fraction, solubilization with guanidine, and renaturation. The characteristics of the binding activity are described and compared with those of authentic viral protein.

Base Sequence↗

PubMed1988

Domain structure of the Moloney murine leukemia virus reverse transcriptase: mutational analysis and separate expression of the DNA polymerase and RNase H activities.

The reverse transcriptase of Moloney murine leukemia virus, like that of all retroviruses, exhibits a DNA polymerase activity capable of synthesis on RNA or DNA templates and an RNase H activity with specificity for RNA in the form of an RNA.DNA hybrid. We have generated a library of linker insertion mutants of the Moloney murine leukemia virus enzyme expressed in bacteria and assayed these mutants for both enzymatic activities. Those mutations affecting the DNA polymerase activity were clustered in the 5'-proximal two-thirds of the gene, and those affecting RNase H were in the remaining 3' one-third. Based on these maps, plasmids were made that expressed each one of the domains separately; assays of the proteins encoded by these plasmids showed that each domain exhibited only the expected activity.

Binding Sites↗

PubMed1986

Analysis of retroviral pol gene products with antisera raised against fusion proteins produced in Escherichia coli.

Portions of the pol gene of Moloney murine leukemia virus (MuLV) were expressed as fusion proteins in Escherichia coli, and the purified proteins were used to elicit antibodies in Escherichia coli, and the purified proteins were used to elicit antibodies in rabbits. The sera were used to examine the mature pol gene products contained in virion particles and identified the reverse transcriptase and a second protein, P46pol, encoded by the 3' portion of the gene. The P46 protein was not phosphorylated and was present at the same molar abundance as the reverse transcriptase. The sera were also used to detect the Pr200gag-pol intracellular precursor protein and to analyze its processing to the mature forms. The proteins formed by several Moloney MuLV mutants were analyzed. Further tests revealed cross-reactivity with Friend MuLV and feline leukemia virus proteins, but not with avian retrovirus proteins.

Cloning, Molecular↗

PubMed1986

Expression of reverse transcriptase activity of human T-lymphotropic virus type III (HTLV-III/LAV) in Escherichia coli.

The pol gene from a biologically active clone of the human T-cell lymphotropic virus type III provirus was inserted into a bacterial expression vector. The resulting gene fusion induced the formation of active reverse transcriptase that could be readily detected in extracts of bacterial cells. The activity exhibited the template and divalent cation requirements of the authentic enzyme. These constructs will be useful for safe and rapid analysis of potential inhibitors of this important enzyme.

DNA, Recombinant↗

PubMed1985

Purification and characterization of murine retroviral reverse transcriptase expressed in Escherichia coli.

Expression of a region of the Moloney murine leukemia virus (M-MuLV) pol gene in Escherichia coli resulted in the synthesis of reverse transcriptase activity which could be detected in crude extracts. Construction of deletions at the 3' terminus of this gene resulted in a 4-fold increase in the level of the reverse transcriptase activity in the soluble fraction of crude lysates and yielded the high level production of a stable protein species of Mr = 71,000. Purification of this protein by column chromatography on DEAE-cellulose, phosphocellulose, polyribocytidylic acid-agarose, and hydroxylapatite indicated that it was a multifunctional enzyme containing RNase H and reverse transcriptase activity. The Mr = 71,000 species had a sedimentation coefficient of 4.65 S by glycerol gradient centrifugation, indicating that the enzyme was a monomer. Using poly(A)+ mRNAs primed with oligo(dT), the enzyme synthesized double-stranded DNA copies between 1.3 and 9.9 kilobases in length. Synthesis of long cDNA required 8 mM Mg2+, 4 mM Mn2+, 2 mM dNTPs, and saturating levels of enzyme. Actinomycin D efficiently limited the enzyme to the first strand synthesis. Additional characteristics of the fusion protein are described.

Chromosome Deletion↗

PubMed1985

Expression of enzymatically active reverse transcriptase in Escherichia coli.

Reverse transcriptase of murine retroviruses is a monomeric protein of approximately 80,000 daltons, which is encoded by the central portion of the viral pol gene. To prepare large quantities of the enzyme, we have constructed gene fusions between the trpE gene and portions of the pol gene of Moloney murine leukemia virus. The inserted pol gene sequences include the entire coding region for the mature enzyme and various amounts of additional coding sequences. Many of these constructs express high levels of reverse transcriptase activity even though the NH2 and COOH termini of the protein product only approximate the correct termini of the authentic protein.

DNA, Recombinant↗

PubMed1983

Complementary DNA sequence of a human cytoplasmic actin. Interspecies divergence of 3' non-coding regions.

We have isolated and sequenced a cloned complementary DNA insert complementary to the messenger RNA of a cytoplasmic actin expressed in human epidermal cells. This provides the first cytoplasmic actin complementary DNA sequence for a vertebrate organism. The actin amino acid sequence predicted from this complementary DNA is identical to that of a bovine cytoplasmic actin and shows 98 and 85% homology with a Dictyostelium and a yeast actin, respectively. The complementary DNA sequence indicates that the 3' end of the mRNA contains an unusually long (greater than 400 nucleotides) 3' non-translated region. A comparison of this 3' non-coding region with those of recently determined actin complementary DNA sequences from other species reveals little or no homology among these sequences. Thus, these results indicate that although the actin amino acid sequences are extremely conserved, the non-coding regions of the mRNAs diverge rapidly.

Actins↗

PubMed1983

The evolution and complexity of the genes encoding the cytoskeletal proteins of human epidermal cells.

In order to explore the differential expression and evolutionary conservation of the genes encoding the cytoskeletal proteins for human epidermal cells, we have constructed a library of bacterial plasmids containing inserts of double-stranded cDNAs complementary to the mRNAs of cultured human epidermal cells. Cloned hybrid plasmids containing 45-95% of the sequences present in keratin, actin, and tubulin mRNAs were isolated and characterized. To identify the cDNAs encoding the four major keratins of human epidermal cells, the clones were initially screened for their ability to hybridize strongly with 32P-labeled cDNA prepared from unfractionated epidermal mRNA (about 30% keratin mRNA). Strongly hybridizing clones were further characterized by positive hybrid selection. Two distinct classes of clones were identified: One class hybridized specifically to the 56 and 58kd keratin mRNAs and one class hybridized specifically to the 46 and 50kd keratin mRNAs. Each class is encoded by a separate multigene family of about 10 genes. The two classes of sequences are conserved throughout vertebrate evolution, indicating the functional importance of each class in filament assembly. Clones containing human cDNA sequences encoding a cytoplasmic actin and alpha-tubulin were selected by hybridization screening using 32P-labeled cloned cDNAs for the mRNAs of beta-actin and alpha-tubulin of embryonic chick brain. The identity of these clones was established by positive hybrid selection and by DNA sequence analysis. Similar to the keratins, the actins and tubulins are also encoded by multigene families which are highly evolutionarily conserved. The availability of cloned cDNAs specific for each of three types of epithelial cytoskeletal proteins allows us to investigate the coordinate expression of their mRNAs during terminal differentiation in human epidermis.

Actins↗

PubMedDate not supplied

Structural requirements for bacterial expression of stable, enzymatically active fusion proteins containing the human immunodeficiency virus reverse transcriptase.

A collection of variant plasmids that express the human immunodeficiency virus (HIV) reverse transcriptase as trpE fusion proteins were generated and scored for their ability to produce stable, active proteins. Trimming portions of the viral pol gene resulted in dramatic increases in yield over earlier constructs; the accumulation of high levels of enzymatically active protein in this system was increased by the retention of the trpE sequences at the amino terminus. A new in situ gel activity assay was used to demonstrate that the major induced protein, containing approximately 68 kD of viral sequences, was the active species.

Cloning, Molecular↗