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PubMed1996

Expression, purification and characterization of recombinant C. Elegans UNC-18.

The Caenorhabditis elegans unc-18-encoded protein (UNC-18) is implicated in the processes of vesicle targeting, docking, and/or fusion. To further characterize the properties of this important neural protein, we expressed it at a high level in Spodoptera frugiperda Sf21 cells using a baculovirus expressing system. A cDNA containing the coding sequence for UNC-18 was inserted into the transfer vector pBlueBac to yield the recombinant virus pAcNPV/unc-18. At maximal expression, the recombinant virus produces a protein of 67 kDa, which constitutes about one-third of total cell protein. The UNC-18 protein was highly purified and its biochemical and functional properties were assessed. The protein is globular with an isoelectric point of 6.95. Circular dichroism spectroscopy indicated that the alpha-helix and beta-sheet account for 10.0 and 59.0%, respectively. Immunolabeling the Sf21 cells expressing UNC-18 showed that the expressed UNC-18 is predominantly localized in the cytoplasm as a soluble monomer. The protein is phosphorylated by protein kinase C and binds to the recombinant C. elegans syntaxin in vitro. These findings suggest that in vesicle traffic UNC-18 is a regulator factor associated with the plasma membrane through syntaxin, although intrinsically cytoplasmic.

Animals↗
PubMed2026

A novel peptide encoded by circTLL1 drives osimertinib resistance in lung cancer by modulating the NT5C2/Ras/PI3K axis.

BACKGROUND: Acquired resistance to osimertinib, a third-generation EGFR tyrosine kinase inhibitor, remains a major clinical challenge in the treatment of non-small cell lung cancer (NSCLC). Although circular RNAs (circRNAs) have been increasingly implicated in drug resistance, most studies have focused on their canonical role as microRNA sponges, while their capacity to encode functional micropeptides remains largely unexplored. This study aimed to identify novel circRNAs involved in osimertinib resistance and to characterize their regulatory functions at the protein level. METHODS: Osimertinib-resistant (OR) NSCLC cell lines were established and validated. High-throughput RNA sequencing was performed to compare the circRNA expression profiles between parental and OR cells. The function of the candidate circRNA was assessed through a series of in vitro and in vivo experiments, including cell viability assays, apoptosis analysis, and xenograft mouse models. Mechanistic investigations involved mass spectrometry, co-immunoprecipitation and western blotting to explore its protein-coding potential and downstream signaling pathways. RESULTS: We identified a novel circRNA, termed circTLL1, that was stably and significantly upregulated in OR-NSCLC cells. Functionally, overexpression of circTLL1 promoted osimertinib resistance, whereas its knockdown restored drug sensitivity both in vitro and in vivo. Mechanistically, we discovered that circTLL1 harbors an open reading frame (ORF) that is translated into a novel 90-amino-acid protein, which we designated circTLL1-90aa. Further investigation revealed that circTLL1-90aa directly interacts with and promotes the degradation of 5'-nucleotidase, cytosolic II (NT5C2), thereby uncoupling nucleotide metabolism from its normal regulatory constraints. The consequent downregulation of NT5C2 leads to elevated GTP levels and leading to the sustained activation of the downstream Ras/PI3K/AKT signaling pathway. CONCLUSION: Our findings unveil a previously unrecognized circRNA/micropeptide/metabolism cascade underlying osimertinib resistance. The identification of the circTLL1-90aa/NT5C2/Ras/PI3K axis not only expands the functional repertoire of the non-coding genome but also provides new insights into the complexity of drug resistance. Given its selective upregulation in resistant cells, circTLL1-90aa holds promise both as a predictive biomarker for treatment stratification and as an actionable therapeutic target, offering a novel strategy to overcome osimertinib resistance in NSCLC patients.

Pyrimidines↗
PubMed1987

Protein IX, a minor component of the human adenovirus capsid, is essential for the packaging of full length genomes.

Human adenovirus type 5 (Ad5) contains a 36-kb double-stranded DNA molecule in an icosahedral capsid. Attempts to construct Ad5 insertion mutants containing DNA of more than about 105% of the genome size resulted in viral progeny in which deletions had occurred suggesting the existence of severe constraints on the size of packageable DNA molecules. To partially circumvent these constraints we used an adenovirus vector, Ad5dlE1,3, with deletions in early regions 1 (E1) and 3 for a total net reduction in genome size of 5349 bp and an expected capacity for inserts of greater than 7 kb. To use this vector efficiently we generated a circular form of dlE1,3 DNA which could be propagated as an infectious bacterial plasmid. When this plasmid was used as a recipient for inserts of various sizes it was found that its capacity was much less than expected and that dlE1,3 virion capsids could not even package DNA as large as the wt genome. Because the E1 deletion of dlE1,3 extends into the coding sequences for protein IX, a minor capsid component known to affect the heat stability of adenovirions, the possibility that absence of this polypeptide might also affect the DNA capacity of the virion was investigated. It was found that when the coding sequences for protein IX were restored the packaging capacity of the vector was also restored to that of wt virions. Thus protein IX is an essential constituent of virion capsids dispensable only for virions containing DNA of less than genomic size.

Adenovirus Early Proteins↗
PubMed1984

Hospital blood bank laboratory data processing system.

A data processing system designed to improve the management and usage of blood and blood products has been developed as a pilot for general application throughout the West Midlands Regional Health Authority. The package provides for the routine clerical, patient record, and stock management aspects of a hospital blood bank laboratory. It can be tailored to the individual requirements of different laboratories and may be implemented in a variety of ways on a family of computer systems of differing power and capacity, thus accommodating widely varying workloads and archiving requirements. Bar code readers are included in the system design, and may be used for data input where standard Codabar labels are affixed to blood products by blood transfusion centres. The system has been installed in one hospital laboratory and preliminary results indicate a high degree of acceptability at all levels. Further refinement of the software to meet minor deficiencies and the requirements of the recent DHSS Health Circular BLD/2/13 are currently being specified.

Blood Banks↗
PubMed1990

Physico-chemical properties of recombinant desulphatohirudin.

Physico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies.

Chemical Phenomena↗
PubMed1981

Nucleotide assignment of alkali-sensitive sites in mouse mitochondrial DNA.

Mature, closed circular mouse mitochondrial DNA contains a significant number of ribonucleotides throughout the genome. Previous studies have implicated the two origins of DNA replication as preferred sites of ribonucleotide retention. We have analyzed the site specificity of ribosubstitution by direct sizing of alkali-treated restriction fragments in comparison with the DNA sequence of untreated restriction fragments of cloned mouse mitochondrial DNA. These results have confirmed the observations that ribonucleotides are retained at the two origins of replication and are most likely remnants of RNA priming events associated with DNA replication. The map location of ribonucleotides at the light strand origin of replication has been refined to a triplet nucleotide (5'-CGG-3') in the light strand initiation region. This approach has demonstrated that all four deoxyribonucleotides are subject to ribosubstitution and no single base (or subset of the four bases) predominates. An examination of selected regions of the mitochondrial DNA genome including the putative coding region for cytochrome oxidase subunit III and regions containing the genes for tRNAPhe, tRNAVal, 12 S rRNA, and 16 S rRNA reveals preferred sites for ribosubstitution. These preferred sites do not relate in any obvious way to the functional aspects of these domains. In addition, the data indicate that every position in the DNA sequences examined can be ribosubstituted at a very low frequency.

Animals↗
PubMed1989

Excision products of immunoglobulin gene rearrangements.

We have isolated circular DNAs from splenocytes of euthymic and athymic mice, and prepared the DNA libraries of 1.5 X 10(6) clones. Hundreds of clones homologous to immunoglobulin (Ig) heavy chain segments (DSP2 and DQ52-JH) or light chain segments (J kappa and J lambda) have been identified. Southern hybridization predicted that three of 12 euthymic mouse clones homologous to DQ52-JH and four of 10 athymic mouse clones homologous to DSP2 contained reciprocal recombination products of D-J joining. Some of these clones were characterized by sequencing: two clones contained the precise excision product of the recombination of a DSP2 segment with either JH2 or JH3 segment; two clones showed imprecise ligation of the DSP2-JH2 coding joint and precise ligation of the DFL16.1-JH3 reciprocal joint in the same molecule. They seem to represent a replacement of the pre-existing DSP2-JH2 rearrangement by joining an upstream DFL16 segment to a downstream JH3 segment. The presence in extrachromosomal DNA of a reciprocal recombination product of DH-JH joining is consistent with the view that immunoglobulin genes, like T cell receptor (TCR) genes, can be rearranged in B cell lineage by the looping-out and excision of chromosomal DNA.

Animals↗
PubMed1988

Structure and organization of Marchantia polymorpha chloroplast genome. I. Cloning and gene identification.

We have determined the complete nucleotide sequence of chloroplast DNA from a liverwort, Marchantia polymorpha, using a clone bank of chloroplast DNA fragments. The circular genome consists of 121,024 base-pairs and includes two large inverted repeats (IRA and IRB, each 10,058 base-pairs), a large single-copy region (LSC, 81,095 base-pairs), and a small single-copy region (SSC, 19,813 base-pairs). The nucleotide sequence was analysed with a computer to deduce the entire gene organization, assuming the universal genetic code and the presence of introns in the coding sequences. We detected 136 possible genes. 103 gene products of which are related to known stable RNA or protein molecules. Stable RNA genes for four species of ribosomal RNA and 32 species of tRNA were located, although one of the tRNA genes may be defective. Twenty genes encoding polypeptides involved in photosynthesis and electron transport were identified by comparison with known chloroplast genes. Twenty-five open reading frames (ORFs) show structural similarities to Escherichia coli RNA polymerase subunits, 19 ribosomal proteins and two related proteins. Seven ORFs are comparable with human mitochondrial NADH dehydrogenase genes. A computer-aided homology search predicted possible chloroplast homologues of bacterial proteins; two ORFs for bacterial 4Fe-4S-type ferredoxin, two for distinct subunits of a protein-dependent transport system, one ORF for a component of nitrogenase, and one for an antenna protein of a light-harvesting complex. The other 33 ORFs, consisting of 29 to 2136 codons, remain to be identified, but some of them seem to be conserved in evolution. Detailed information on gene identification is presented in the accompanying papers. We postulated that there were 22 introns in 20 genes (8 tRNA genes and 12 ORFs), which may be classified into the groups I and II found in fungal mitochondrial genes. The structural gene for ribosomal protein S12 is trans-split on the opposite DNA strand. The universal genetic code was confirmed by the substitution pattern of simultaneous codons, and by possible codon recognition of the chloroplast-encoded tRNA molecules, assuming no importation of tRNA molecules from the cytoplasm. The nucleotide residue A or T is preferred at the third position of the codons (G+C, 11.9%) and in intergenic spacers (G+C, 19.5%), resulting in an overall G+C content that is low (28.8%) throughout the liverwort chloroplast genome. Possible gene expression signals such as promoters and terminators for transcription, predicted locations of gene products, and DNA replicative origins are discussed.

Base Sequence↗
PubMed2000

Borrelia genomes in the year 2000.

All analyzed members of the spirochete genus Borrelia contain a linear chromosome about 910 kbp long. The complete sequence of the B. burgdorferi B31 genome predicts that its chromosome carries essentially all of this organism's housekeeping genes. In accordance with these bacterial species' obligatory parasitic lifestyle, its genes encode enzymes that are capable of only a minimal metabolism, in which all nucleotides, amino acids, fatty acids and enzyme cofactors must be scavenged from the host. In addition to the chromosome, all Borrelia isolates examined carry multiple linear and circular plasmids with lengths between 5 and 200 kbp. The plasmids, which account for over 600 kbp in isolate B31, carry very few genes with homology to genes outside of the Borrelia genus. But they do carry numerous predicted lipoprotein genes, many of which are have been shown to be or are expected to be outer surface proteins. Ten of the linear plasmids have strikingly low protein coding potential for bacterial DNA. These plasmids have enjoyed numerous past duplicative rearrangements, which have resulted in the presence of a substantial fraction of the DNA that appears to be currently undergoing mutational decay, presumably because it is no longer under selection for function.

Borrelia↗
PubMed1989

Expression of native rabbit light meromyosin in Escherichia coli. Observation of a powerful internal translation initiation site.

The cDNA-sequence coding for rabbit skeletal muscle light meromyosin (LMM) was placed under the control of the lambda promoter (PL) of an Escherichia coli expression vector. The resulting plasmid pEXLMM74 expressed non-fused rabbit skeletal muscle LMM with yields ranging from 1 to 5% of the total proteins of E. coli. This LMM was specifically recognized by polyclonal antibodies raised against chicken pectoralis muscle myosin. It could be highly enriched from E. coli extracts by using two cycles of high and low ionic strength buffer. The partially purified protein contained a major side-product, with a calculated molecular mass of 59 kilodaltons, that is produced by translation initiation from a site in the coding region of LMM. After deletion of the translation initiation site derived from the expression plasmid, only the 59 kilodalton protein is expressed in E. coli from the resulting plasmid pEXLMM59. Both the 74 and 59 kilodalton proteins were shown to form paracrystals. They were studied by electron microscopy using negative staining and were found to show characteristic striations with an axial periodicity of about 43 nm. By circular dichroism measurement we showed that the purified 59 kilodalton protein is folded mostly as an alpha-helix.

Animals↗
PubMed2002

Characterization of a novel isoform of alpha-nascent polypeptide-associated complex as IgE-defined autoantigen.

The nascent polypeptide-associated complex is required for intracellular translocation of newly synthesized polypeptides in eukaryotic cells. It may also act as a transcriptional coactivator in humans and various eukaryotic organisms and binds to nucleic acids. Recently, we provided evidence that a component of nascent polypeptide-associated complex, alpha-nascent polypeptide-associated complex, represents an IgE-reactive autoantigen for atopic dermatitis patients. By oligonucleotide screening we isolated a complete cDNA coding for a so far unknown alpha-nascent polypeptide-associated complex isoform from a human epithelial cDNA library. Southern blot hybridization experiments provided further evidence that alpha-nascent polypeptide-associated complex is encoded by a gene family. Recombinant alpha-nascent polypeptide-associated complex was expressed in Escherichia coli as a soluble, His-tagged protein, and purified via nickel affinity chromatography. By circular dichroism analysis it is demonstrated that purified recombinant alpha-nascent polypeptide-associated complex represents a folded protein of mixed alpha-helical and beta-sheet conformation with unusual high thermal stability and remarkable refolding capacity. Complete recombinant alpha-nascent polypeptide-associated complex (215 amino acids) and its 86 amino acid C-terminal fragment specifically bound IgE autoantibodies. Recombinant alpha-nascent polypeptide-associated complex also inhibited IgE binding to natural alpha-nascent polypeptide-associated complex, demonstrating the presence of common IgE epitopes between the recombinant and natural protein. Furthermore, recombinant alpha-nascent polypeptide-associated complex induced specific lymphoproliferative responses in peripheral blood mononuclear cells of a sensitized atopic dermatitis patient. As has been proposed for environmental allergens it is possible that T cell responses to IgE-defined autoantigens may contribute to the chronic skin manifestations in atopic dermatitis.

Amino Acid Sequence↗
PubMed1996

Characterization of the Lymantria dispar nucleopolyhedrovirus 25K FP gene.

The Lymantria dispar nucleopolyhedrovirus (LdMNPV) gene encoding the 25K FP protein has been cloned and sequenced. The 25K FP gene codes for a 217 amino acid protein with a predicted molecular mass of 24870 Da. Expression of the 25K FP protein in a rabbit reticulocyte system generated a 27 kDa protein, in close agreement with the molecular mass predicted from the nucleotide sequence. The gene is located between 40.3 and 40.8 map units on the viral genome. It is transcribed in a counterclockwise direction with respect to the circular map at late times during the infection cycle from a consensus baculovirus late promoter. The LdMNPV and Autographa californica nucleopolyhedrovirus (AcMNPV) 25K FP proteins exhibit 52% amino acid identity with several regions showing greater than 75% identity. Homologues to the AcMNPV orf59 and orf60 were also identified upstream (with respect to the genome) of the 25K FP gene in LdMNPV and exhibit 52% and 45% amino acid identity, respectively.

Amino Acid Sequence↗
PubMed1985

Complete nucleotide sequence of the Drosophila transposable element copia: homology between copia and retroviral proteins.

We have determined the complete nucleotide sequence of the copia element present at the white-apricot allele of the white locus in Drosophila melanogaster. This transposable element is 5,146 nucleotides long and contains a single long open reading frame of 4,227 nucleotides. Analysis of the coding potential of the large open reading frame, which appears to encode a polyprotein, revealed weak homology to a number of retroviral proteins, including a protease, nucleic acid-binding protein, and reverse transcriptase. Better homology existed between another part of the copia open reading frame and a region of the retroviral pol gene recently shown to be distinct from reverse transcriptase and required for the integration of circular DNA forms of the retroviral genome to form proviruses. Comparison of the copia sequence with those of the Saccharomyces cerevisiae transposable element Ty, several vertebrate retroviruses, and the D. melanogaster copia-like element 17.6 showed that Ty was most similar to copia, sharing amino acid sequence homology and organizational features not found in the other genetic elements.

Amino Acid Sequence↗
PubMed1993

A tRNA(Val) (GAC) gene of chloroplast origin in sunflower mitochondria is not transcribed.

A tRNA(Val) (GAC) gene is located in opposite orientation 552 nucleotides (nt) down-stream of the cytochrome oxidase subunit III (coxIII) gene in sunflower mitochondria. The comparison with the homologous chloroplast DNA revealed that the tRNA(Val) gene is part of a 417 nucleotides DNA insertion of chloroplast origin in the mitochondrial genome. No tRNA(Val) is encoded in monocot mitochondrial DNA (mtDNA), whereas two tRNA(Val) species are coded for by potato mtDNA. The mitochondrial genomes of different plant species thus seem to encode unique sets of tRNAs and must thus be competent in importing the missing differing sets of tRNAs.

Base Sequence↗
PubMed1999

Strand asymmetry and codon usage bias in the chloroplast genome of Euglena gracilis.

It is shown that the two strands of the chloroplast genome from Euglena gracilis are asymmetric with regards to nucleotide composition. This asymmetry switches at both the origin of replication and a location that is halfway around the circular genome from the origin. In both halves of the genome the leading strand is G+T-rich, having a bias toward G over C and T over A, and the lagging strand is A+C-rich. This asymmetry is probably the result of a difference in mutation dynamics between the leading and lagging strands. In addition to composition asymmetry, the two strands differ with regards to coding content. In both halves of the genome the vast majority of genes are coded by the leading strand. These two aspects of strand asymmetry are then applied to a statistical test for selection on codon usage. The results indicate that selection on codon usage is limited to genes on the leading strand; no gene on the A+C-rich lagging strand shows evidence for selection, suggesting that highly expressed genes are coded predominantly on the strand of DNA that is the leading strand during replication. On the basis of these observations it is proposed that the coding strand bias is generated by selection to code highly expressed genes on the leading strand to coordinate the direction of replication and transcription, thereby increasing the potential rate of both reactions.

Animals↗
PubMed1983

[TEM-type beta-lactamase coded by the plasmid from Citrobacter sp].

beta-Lactamase was isolated from the cells of E. coli, strain 1039, a transconjugant carrying the plasmid first detected in Citrobacter sp. beta-Lactamase was purified to obtain a homogeneous preparation. The activity of the enzyme was estimated by a modification of the potentiometric method providing determination of up to 0.1 mumol of penicilloinic acid. The activity of the pure enzyme was 206 mumol per 1 mg of protein per minute. The molecular weight determined by gel filtration was 21500. The substrate profile of beta-lactamase corresponded to the TEM type. Preliminary incubation with methicillin resulted in a significant decrease in the enzyme activity. Carbenicillin, dicloxacillin, oxacillin and cephalothin induced no decrease in the activity of the enzyme when subjected to preliminary incubation with it. The use of the functional group reagents allowed detecting residues of serine and lysine in the enzyme active centre or close to it. Km and Kcat for some antibiotics were evaluated. The ratio of alpha- and beta-structures in the molecule of beta-lactamase was determined with the method of circular dichroism (CD). The fraction of the alpha-spiral areas amounted to 30 +/- 5 per cent and the fraction of beta-structures amounted to 20 +/- 5 per cent. On attachment of methicillin to the molecule of the enzyme the ratio of alpha- and beta-structures did not change which may be considered as a preliminary indication of the centre "immobility" in the process of catalysis. Slow hydroysis of methicillin by beta-lactamase was shown with the method of CD.

Chemical Phenomena↗
PubMed1977

Synthesis of phiX174 viral DNA in vitro depends on phiX replicative form DNA.

A cell-free system that catalyzes phiX174 replicative form I (supercoiled circular duplex, RFI)-dependent phiX174 DNA synthesis has been isolated from Escherichia coli infected with phiX174 phage. The products formed with such preparations are viral strands as judged by hybridization to poly(U,G) followed by equilibrium centrifugation in CsCl. This phiX174 DNA-synthesizing involves formation of DNA-protein complexes that sediment in neutral sucrose with S values of 50, 60-70, and higher. The 50S complex contained a rolling-circle replicative intermediate DNA with an extended tail of single-stranded viral DNA. The DNA contained in the 60-70S region was a mixture of circular and linear single-stranded DNA, RFI, and RFII with an extended single-stranded tail. Such complexes have been isolated during in vivo progeny phiX174 DNA synthesis [Fujisawa, H. & Hayashi, M. (1976) J. Vriol. 19,409]. In vitro, maximal phiX174 DNA synthesis was shown to require the genetically defined proteins E. coli dna B, dna C, dna G, dna Z, rep. phiX174 gene A product, and other phiX174 coded proteins. The synthesis of phiX174 DNA is ATP-dependent and is inhibited by nalidixic acid and novobiocin but is resistant to rifampicin.

Chemical Phenomena↗
PubMed1985

Introduction of mouse C epsilon genes into Cos-7 cells and fertilized mouse eggs.

A fragment of the cloned gene for the mouse C epsilon chain, coding for the first, second, third, and fourth domains, has been coupled to the SV40 promotor region (pSV2-mC epsilon). About 50 copies of pSV2-mC epsilon or its PvuII-EcoRI fragments were introduced into Cos-7 cells. Expression of PvuII-EcoRI fragments of pSV2-mC epsilon was observed in about 50% of the Cos-7 cells by indirect fluorescence staining. However, no expression of circular pSV2-mC epsilon was observed. About 200 copies of linearized pSV2-mC epsilon with EcoRI were introduced into fertilized mouse eggs. Two of 78 mice born from these eggs had integrated mouse C epsilon genes. Mouse C epsilon gene was shown to be integrated in a tandem array and as intact structures without undergoing gross deletions or rearrangements, judged from the Southern blotting patterns from several restriction enzymes. The first transgenic mouse was mated to a normal male to examine whether mouse C epsilon gene were stably transmitted to progeny. Among 5 mice to which the C epsilon gene had been transmitted, one deleted 5 copies of this gene and another deleted one junction fragment, thus demonstrating relatively unstable transmission. No C epsilon mRNA was detected in the liver, kidney, brain, lung, skeletal muscle, heart, testis, or spleen of a transgenic mouse.

Animals↗