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PubMed1977

Elongation factor Tu resistant to kirromycin in an Escherichia coli mutant altered in both tuf genes.

A mutant of Escherichia coli is described that displays kirromycin resistance in a cell-free system by virtue of an altered elongation factor Tu (EF-Tu). In poly(U)-directed poly(Phe) synthesis the kirromycin resistance of the crystallized enzyme ranged between a factor of 80 and 700, depending on temperature. Similarly, kirromycin-induced EF-Tu GTPase activity uncoupled from ribosomes and aminoacyl-tRNA required correspondingly higher concentrations of the antibiotic. Resistance of EF-Tu to kirromycin is a consequence of a modified enzyme structure as indicated by its altered fingerprint pattern.P1 transduction experiments showed that the kirromycin-resistant EF-Tu is coded by an altered tufB gene (tufB1). The known existence of two genes coding for EF-Tu would interfere with the recognition of a mutant altered in only one of those genes, if the mutation were recessive. Because kirromycin blocks EF-Tu release from the ribosome, kirromycin sensitivity is dominant, as shown by the failure of a mixed EF-Tu population to express resistance in vitro. Therefore, phenotypic expression of kirromycin resistance in vivo appears to be only possible if the EF-Tu mutant lacks an active tufA gene, a property likely to be inherited from the parental D22 strain. The observations that introduction of a tufA(+) region makes the resistant strain sensitive to the antibiotic and that transduction of tufB1 into a recipient other than E. coli D22 yields kirromycin-sensitive progeny support these conclusions.

Anti-Bacterial Agents↗

PubMed1986

A chimeric plasmid from cDNA clones of poliovirus and coxsackievirus produces a recombinant virus that is temperature-sensitive.

We have inserted a 405-nucleotide fragment from the 5' noncoding region of the coxsackievirus B3 genome into an infectious cDNA copy of the poliovirus RNA genome. Transfection of plasmid DNA containing this hybrid genome construct into cultured monkey cells produced infectious virus. Recombinant virus stocks displayed a temperature-sensitive phenotype for growth at 37 degrees C. We found that there is a dramatic reduction in the level of viral proteins and viral RNAs in HeLa cells infected with the recombinant at 37 degrees C compared to that obtained at 33.5 degrees C. Thus, insertion of a portion of the coxsackievirus genome into the poliovirus genome produces a temperature-sensitive recombinant virus. That this substitution occurs in a region of the poliovirus genome that, to date, has not been shown to have any coding function suggests that RNA sequences involved in replicase recognition or ribosome binding may contribute to the temperature-sensitive phenotype of the recombinant virus.

Animals↗

PubMed1978

Major transplantation antigens in host responses to infection.

There is increasing support for the concept that in all of their varied immunologic functions, T lymphocytes act through a dual specificity. In the case of intracellular viral infections, the recognition required for cytolysis involves both a "self" component, coded for by the genes that specify the major transplantation antigens, and an "X" component, provided by viral antigens expressed on the target-cell surface.

Animals↗

PubMed1986

A general method for retrieving the components of a genetically engineered fusion protein.

Escherichia coli expression vectors encoding an acid-labile aspartyl-proline (Asp-Pro) dipeptide bridging two protein sequences were constructed and used to synthesize two different bovine growth hormone (bGH) fusion proteins. The codons GAT-CCX coding for Asp-Pro are provided by the recognition site for Bam HI (GGATCC). Treatment of the bGH fusion proteins at low pH in the presence of guanidine hydrochloride releases the bGH moiety from the fusion protein. The release of the bGH from the fusion protein specifically requires the Asp-Pro dipeptide linking the bGH sequence to the fusion protein. The bGH released from the fusion protein retains anti-bGH immunoreactivity as well as the ability to bind to growth hormone receptor in vitro.

Acids↗

PubMed1990

Loss of human CR1- and murine Crry-like exons in human CR2 transcripts due to CR2 gene mutations.

Analysis of the coding sequences of the murine Cr2 gene indicated that it contains two distinct regions of homology to the murine Crry, and human CR1 and CR2 genes. The last 15 short consensus repeats (SCR) of Cr2 are very similar to the 15 reported SCR of human CR2. These 15 SCR of Cr2, plus the transmembrane and cytoplasmic domains, make up a protein very similar in organization and sequence to the human CR2 gene product. Another Cr2 transcript contains the sequences encoding the previously described 15 SCR plus those encoding another six amino terminal SCR. These amino terminal SCR are very similar to those of Crry and the first six SCR seen within the CR1 long homologous repeats. Amino acid sequence similarity analysis, however, indicated that the sequences encoding the six amino terminal SCR of Cr2 evolved from a separate lineage of SCR than Crry and CR1, suggesting that the human counterparts to these additional Cr2 SCR had not been identified. Using the cDNA sequences specific for the amino terminal SCR of murine Cr2, the human counterparts were isolated and localized within the human CR2 gene between those nucleotides encoding the signal sequence and those encoding the first SCR of the mature human CR2 protein. Unlike the murine Cr2 gene products, these Crry/CR1-like sequences of the CR2 gene are not maintained in mature CR2 mRNA, and thus represent pseudoexons. DNA sequence analysis of the pseudoexon homologous to that encoding the first SCR of the murine Cr2 gene indicated that a stop codon has been introduced within the human coding sequence but that the 5' and 3' splice recognition sequences appear to be functional. Although the stop codon would block the translation of a transcript with this exon, it would not inhibit the incorporation of the exon within the mature CR2 transcript. Therefore, another mutation must have been introduced within the human CR2 gene such that both this exon and the other CR1/Crry-like exons are removed from mature CR2 transcripts.

Amino Acid Sequence↗

PubMed1976

Blocking of MLC stimulation by anti-Ia sera: studies with the virus plaque assay.

The development of congenic mouse strains identical at the H-2K and H-2D loci but differing by I-region associated (Ia) determinants has permitted an association to be established between Ia determinants and stimulation in mixed lymphocyte culture reactions (MLR). The present experiments were undertaken to establish whether the Ir-coded control of MLR operated at the level of recognition or of stimulation. Reciprocal MLR were established between A.TH and A.TL mouse spleen cells in the presence or absence of anti-Ia sera directed either at determinants of the stimulating or responding cells. The number of T cells responding was assessed by the virus plaque assay. Anti-Ia sera directed against the responding cells were no more inhibitory of the MLR than normal mouse serum. In contrast, anti-Ia sera directed against determinants of the mitomycin-treated stimulating cells markedly inhibited activation of T cells in the MLR.

Animals↗

PubMed1992

Expression and purification of the cytoplasmic tail of an endocytic receptor by fusion to a carbohydrate-recognition domain.

Gene fusion has been used to produce the cytoplasmic domain of an endocytic receptor. DNA sequences coding for the 52 COOH-terminal amino acids of the mannose receptor from human macrophages, including the 41-amino acid cytoplasmic tail, were fused to the codons specifying the carbohydrate-recognition domain (CRD) of rat mannose-binding protein. The fusion protein was expressed in Escherichia coli and purified in one step on mannose-Sepharose, making use of the carbohydrate-binding activity of the CRD. The tail peptide was released from the fusion protein using endoproteinase Arg-C. This method provides an alternative to chemical synthesis for the production of midlength peptides.

Amino Acid Sequence↗

PubMed1980

Functional and molecular organisation of an antigen-specific suppressor factor from a T-cell hybridoma.

Thymus-dependent (T) lymphocytes have been shown to have antigen specificity. The antigen receptor on T lymphocytes, in contrast to that on B lymphocytes, does not appear to be of the conventional immunoglobulin (Ig) type. Studies on the antigen-specific factors derived from helper and suppressor T cells (Ts) demonstrated that they possess determinants with antigen binding affinity and products of genes in the H-2 complex (MHC). Furthermore, antibodies against the variable region of Ig heavy chains or idiotypes have been shown to react with T-cell antigen receptors as well as antigen-specific helper and suppressor T-cell factors (TsF). It is, therefore, conceivable that at least two gene products are involved in the structural entity of these receptors: one each coded for by genes in either. To establish the molecular nature of the recognition component of T cells we have used homogeneous TsF from a T-cell hybridoma with a specific function. We report here that the antigen binding and I-J coded molecules on TsF are independently synthesised in the cytoplasm, and are secreted as an associated form of the two molecules; this association is required for antigen-specific suppression of antibody response.

Animals↗

PubMed1983

The ethically trained physician: myth or reality?

Through a questionnaire distributed to 300 physicians in the Toronto area, three aspects of their ethical awareness were examined: the formal codes, the need for consultation in making decisions and the need for training in medical ethics. Most of the physicians (81%) felt that they were facing ethical problems in their daily practice. A majority of these would try to solve the problems either themselves (30%) or through discussion with a colleague (43%). When they turned outside the profession it was sometimes to a lawyer (12%), which suggests concern with the legalities of some situations. Only a small proportion of the respondents were found to be familiar with two of the established codes of ethics, yet 13% would still turn to the code of the Canadian Medical Association for guidance. Finally, there was widespread recognition of the need for proper training in medical ethics and for the establishment of a specialty in this field.

Attitude of Health Personnel↗

PubMed1990

Molecular structure, chromosome assignment, and promoter organization of the human matrix Gla protein gene.

Matrix Gla protein (MGP) is an 84-residue vitamin K-dependent protein initially isolated from bovine bone. MGP is also expressed at high levels in heart, kidney, and lung and is up-regulated by vitamin D in bone cells. To characterize the genomic sequences responsible for the regulated expression of this gene, we screened a human genomic library using a MGP cDNA probe and obtained two clones containing the MGP locus. The human MGP gene spans 3.9 kilobases of chromosomal DNA and consists of four exons separated by three large intervening sequences which account for more than 80% of the gene. Southern analysis of total human genomic DNA indicated the presence of a single copy of the MGP gene. Hybridization of the hMGP cDNA to a series of Chinese Hamster x human hybrid clones assigned this gene to the short arm of the human chromosome 12 (12p). The N-terminal sequences of the known vitamin K-dependent vertebrate proteins reveal a transmembrane signal peptide, followed by a putative gamma-carboxylation recognition site and a Gla-containing domain. Each of these regions correspond to a separate exon in MGP. MGP also contains a fourth exon of unknown function which codes for 11 residues and lies between the transmembrane signal peptide and the putative recognition site for the gamma-carboxylase. This four-exon organization is essentially identical to that of bone Gla protein and is quite different from the two exon organization encoding this region in the other known vitamin K-dependent proteins. Analysis of the MGP gene promoter revealed, in addition to the typical TATA and CAT boxes, the presence of a number of putative regulatory sequences homologous to previously identified hormone and transcription factor responsive elements. In particular, two regions of the promoter were delineated containing possible binding sites for retinoic acid and vitamin D receptors.

Amino Acid Sequence↗

PubMed1980

Isolation and sequence of the gene for iso-2-cytochrome c in Saccharomyces cerevisiae.

The two apocytochrome c proteins of yeast are coded for by separate genes. Iso-2-cytochrome c differs from the iso-1 protein at 17 positions within a homologous sequence of 108 amino acids. The previously cloned iso-1-cytochrome c coding sequence has been used to identify lambda-yeast recombinant phage containing the gene for iso-2-cytochrome c. The latter protein contains the dipeptide Ala-Ala which is coded for by the nucleic acid sequence G-C-N-G-C-N. The recognition specificity of restriction endonuclease Fnu4HI for G-C-N-G-C provided a rapid means of locating the region of the cloned fragment which codes for iso-2-cytochrome c. The DNA sequence of this gene has been determined and compared with that of the iso-1-cytochrome c locus. There is no intervening sequence within the gene for iso-2-cytochrome c. At 45 of the 91 positions for which iso-1- and iso-2-cytochrome c have the same amino acid, the codons differ. Such third position variation does not occur within the region coding for amino acids 70-80, the protein sequence that is also most conserved among all eukaryotic cytochromes c.

Apoproteins↗

PubMed1991

Construction of cloning cartridges for development of expression vectors in gram-negative bacteria.

A cloning cartridge was constructed that can be inserted into a plasmid of choice to form an expression vector in which gene expression is inducible with an inexpensive inducer, sodium salicylate, at low concentrations. This cartridge consists of a 3.6-kb restriction fragment which contains the positive regulatory gene nahR from plasmid NAH7, a promoter, PG, that nahR regulates, a multiple cloning site, a transcription terminator, and a gene conferring tetracycline resistance. Within promoter PG of the cloning cartridge, a sequence of three nucleotides upstream of the ATG sequence encoding the initiation codon was altered to create an NdeI recognition site (CATATG) for cloning of the 5' end of a gene without affecting the distance between the transcription start site and the gene coding region. In addition, the 5' end of a gene can be converted into an NdeI recognition site without altering the amino acid sequence it encodes and then cloned into this cartridge for regulated expression. Several other synthetic restriction sites were also inserted downstream of the NdeI site for accepting the 3' end of a cloned gene. A derivative of this cloning cartridge lacking the NdeI sequence was also constructed for cloning and expression of a restriction fragment containing a gene(s) of unknown sequence. Use of the cloning cartridges in a broad-host-range plasmid has allowed successful cloning and inducible expression of several genes in all of the gram-negative bacterial tested to date. Protein production to at least 10% of the total soluble cell proteins was observed from a cloned gene expressed in Pseudomonas putida.

Bacterial Proteins↗

PubMed2026

VIPR RNA-guided DNA recognition by noncontiguous geometric triplex formation.

Viral interference programmable repeat (VIPR) systems use a noncontiguous code for RNA-guided transcriptional silencing. How the Vipr protein and a VIPR RNA (vrRNA) comprising alternating GGY and NN segments achieve precise DNA targeting is unknown. Here, we present 21 cryo-electron microscopy structures that help explain the mechanism of target engagement. Vipr protomers oligomerize along the vrRNA to form a right-handed helical filament, sequestering each GGY motif and positioning the adjacent NN bases for target base pairing. DNA binding, in which every third nucleotide is skipped, results in a gapped vrRNA-DNA hybrid helix that encircles the nontarget DNA strand to form a geometric triplex. These findings suggest that triplex-mediated target-strand handoff could enable noncontiguous and programmable RNA-guided DNA recognition in VIPR systems.

DNA↗

PubMed1987

Recognition of hierarchically encoded images by technical and biological systems.

All the contours and regions of objects can be mapped to code-trees of the Hierarchical Structure Code (HSC). Invariant features like structure classes, shape descriptions, or relations between structures and components may be easily extracted from the HSC. HSC-based pattern recognition provides a straightforward transition between the signal-space of the image and the space of its symbolic representation. Physiological data are well predicted and do not exclude an implementation of an HSC-based system within the visual cortex.

Cybernetics↗

PubMed1984

Signal recognition particle triggers the translocation of storage globulin polypeptides from field beans (Vicia faba L.) across mammalian endoplasmic reticulum membrane.

Hybridization-selected mRNAs coding for individual storage globulin polypeptides of field beans (Vicia faba L.) were translated in a cell-free system. Added mammalian signal recognition particle (SRP) recognizes cleavable signal peptides of the major vicilin and both legumin polypeptide precursors and induces translational arrest. The latter can be released by potassium-washed membranes (K-RM) leading to shortened polypeptides protected against proteases. Thus, SRP and K-RM function in a similar way with plant polypeptides as described for mammalian secretory proteins [(1981) J. Cell Biol. 91, 557-561]. Obviously, the initial steps in the biosynthesis and processing of plant storage globulin polypeptides are principally identical to those of animal secretory proteins.

Animals↗

PubMed1976

A cybernetic approach to the origin of the genetic coding mechanism. II. Formation of the code series.

The sequential fulfillment of the principle of succession necessarily guides the main steps of the genetic code evolution to be reflected in its structure. The general scheme of the code series formation is proposed basing on the idea of "group coding" (Woese, 1970). The genetic code supposedly evolved by means of successive divergence of pra-ARS's loci, accompanied by increasing specification of recognition capacity of amino acids and triplets. The sense of codons had not been changed on any step of stochastic code evolution. The formulated rules for code series formation produce a code version, similar to the contemporary one. Based on these rules the scheme of pra-ARS's divergence is proposed resulting in the grouping of amino acids by their polarity and size. Later steps in the evolution of the genetic code were probably based on more detailed features of the amino acids (for example, on their functional similarities like their interchangeabilities in isofunctional proteins).

Amino Acids↗

PubMed1985

Naturally occurring BK virus variants (JL and Dik) with deletions in the putative early enhancer-promoter sequences.

The genomes of two independently isolated BK virus (BKV) variants (JL and Dik) were compared with prototype BKV DNA by restriction endonuclease mapping and sequence analysis. Differences were mainly detected in two regions: the BKV (JL) and BKV (Dik) putative early enhancer-promoter regions and the middle of the T-antigen-coding regions. Base sequence analysis of these two regions showed the following. (i) The putative enhancer-promoter regions of BKV (Dik) and BKV (JL) contained only one 68-base-pair (bp) unit of the 68-bp triplication (the central copy of which is missing 18 bp) present in prototype BKV. (ii) In the same region, BKV (JL) and BKV (Dik) contained unique stretches of DNA 33 and 63 bp long, respectively. In these 63 bp, a sequence which was very similar to the proposed simian virus 40 enhancer core sequence (GGAGTGGAAAG) was present. (iii) The altered restriction endonuclease recognition sites in the sequenced part of the T-antigen-coding region of BKV (JL) and BKV (Dik) were due to base sequence changes, leaving the amino acid sequence unchanged.

BK Virus↗

PubMedDate not supplied

The cyanobacteriales: a legitimate order based on the type strain Cyanobacterium stanieri?

As a logical consequence of the definition of a bacterium (Stanier and van Niel, 1962), R. Y. Stanier created the name "cyanobacteria" as a replacement for "blue-green algae". As such, cyanobacteria entered the 8th issue of Bergey's Manual of Determinative Bacteriology 1974 as members of the Procaryotae Murray 1968, this kingdom being composed of two divisions, Cyanobacteria and Bacteria. An even tighter integration of cyanobacteria with other bacteria was proposed by Gibbons and Murray (1978) for the next edition of Bergey's Manual. These authors suggested that the cyanobacteria be integrated as an order Cyanobacteriales in the class Photobacteria. However, this proposal was doomed to failure by constraints imposed under present rules of the Bacteriological Code (Lapage et al., 1976), one of which is that the type of an order is the genus upon whose name the higher taxon is based. A genus Cyanobacterium did not exist when Gibbons and Murray made their proposal, and a subsequent special request by the same authors for an exemption from this rule was not granted (Judicial Commission of the International Committee on Systematic Bacteriology, Holt, 1978). We present here a revised classification for unicellular cyanobacteria dividing in one plane wherein we propose, among other changes, the creation of two new genera. Cyanobium and Cyanobacterium. With the creation of the latter genus, the requirement for recognition of cyanobacteria as a legal order Cyanobacteriales under the Bacteriological Code should be fulfilled. We suggest that the type species of this genus by Cyanobacterium stanieri, in honor of the late Roger Y. Stanier.

Base Composition↗