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Biomedical subjects

J Carey

Publications and source records attributed to J Carey.

At least 127 records · Page 7Linked to original sources
PubMed1985

Translation activates the paused transcription complex and restores transcription of the trp operon leader region.

It has been proposed that RNA polymerase pausing in the leader region of the tryptophan (trp) operon of Escherichia coli is responsible for the synchronization of transcription and translation essential to attenuation control. In this report we use an in vitro coupled transcription/translation system to study the effect of trp leader peptide synthesis on RNA polymerase pausing in the trp leader region. Wild-type and translation-defective trp leader templates of E. coli and Serratia marcescens were employed, and pause RNA synthesis and paused complex release (activation) were quantified relative to synthesis of the terminated leader transcript. It was observed that pausing in the trp leader region was prolonged when translation of the leader transcript was reduced by mutations in the leader region or by addition of the translation inhibitor kasugamycin or chloramphenicol. Experiments with S-30 extracts from a mutant strain that is inefficient in translating the tryptophan codons in the leader transcript indicated that ribosome movement to these codons also releases the paused transcription complex. These findings indicate that the paused trp leader transcription complex resumes transcription when released by ribosome movement over the leader peptide coding region. This release would facilitate the coupling of transcription and translation essential to attenuation control.

Amino Acid Sequence↗
PubMed1984

Failure of translational repression in the phage f2 op3 mutant is not due to an altered coat protein-RNA interaction.

A secondary phenotype of the op3 mutant of RNA bacteriophage f2 is the absence of translational repression of the phage replicase gene by the phage coat protein. We have synthesized RNA fragments corresponding to the site of translational repression for both the wild type and the op3 mutant. Using a quantitative assay, we show that the affinity of the closely related R17 coat protein for the mutant and wild type RNA fragments is the same. In addition, we find that the op3 and R17 coat proteins bind to the wild type RNA fragment with essentially identical dissociation constants. Thus, the altered regulation of replicase protein synthesis in the op3 mutant does not appear to be due simply to a reduced affinity of the translational repressor for its target site.

Bacteriophages↗
PubMed1984

Parietal foramina clavicular hypoplasia. An autosomal dominant syndrome.

A male infant was evaluated with macrocephaly, scaphocephaly , a high forehead, a parietal foramen, a midline posterior occipital dermoid, and sloped shoulders. A skeletal survey also showed distal hypoplasia of the clavicles with bilateral loss of the acromion. Similar features were evident in his father, grandfather, and two paternal great-aunts. To our knowledge, this is the second report of this syndrome, which may be designated as parietal foramina-cleidocranial dysplasia. Our three-generation family and male-to-male transmission clearly established an autosomal dominant mode of inheritance for this syndrome.

Adult↗
PubMed1984

Dominant optic atrophy, deafness, ptosis, ophthalmoplegia, dystaxia, and myopathy. A new syndrome.

Twenty-three members of a 96-member family exhibited an autosomal dominant disorder which has not previously been described. This disorder involves progressive optic atrophy, abnormal electroretinography without retinal pigment changes, and progressive sensorineural hearing loss usually evident in the first or second decade of life. In midlife, ptosis, ophthalmoplegia, dystaxia, and a nonspecific myopathy occur.

Adult↗
PubMed1984

Isolation and sequencing of a cDNA clone for a human HLA-ABC antigen.

A cDNA clone, pHLA-A, containing sequences specific for a human class I HLA antigen heavy chain, was isolated from a bank of clones made from partially purified HLA-ABC heavy chain mRNA from the human lymphoblastoid cell line Bristol 8 (HLA-A1, A2, B8, B16). The clone corresponded to sequence for the -COOH-terminal 117 amino acids of an HLA-ABC alpha-chain. It differed in at least 15 positions from the published HLA-B7 amino acid sequence but in only two residues when compared to the partial HLA-A2 protein sequence, and was identical to the HLA-A3 protein sequence derived from the nucleotide sequence of the gene. It also differed from some published human HLA-ABC sequences by the addition of three extra -COOH-terminal amino acids: cysteine-lysine-valine. The clone may correspond to either the HLA-A1 allele, for which independent sequence information is not available, or to HLA-A2, in which case there are possible explanations for the discrepancies. Comparison of the pHLA-A sequence with genomic HLA sequences suggests variations in splicing at the end of the protein coding region in some HLA-ABC heavy chain genes, and the use of alternative poly(A) addition sites.

Amino Acid Sequence↗
PubMed1983

Interaction of R17 coat protein with synthetic variants of its ribonucleic acid binding site.

The specificity of the interaction between R17 coat protein and its site of translational repression on R17 RNA was studied by enzymatically synthesizing 23 sequence variants of the RNA binding site and measuring their affinity to the coat protein by a nitrocellulose filter binding assay. Experiments using oligomers truncated on the 3' and 5' termini allowed precise determination of the edges of the binding domain. Several oligomers which disrupted one or more of the base pairs in the binding site failed to bind coat protein, establishing the importance of RNA secondary structure for the interaction. Substitution at two single-stranded positions with each of the common bases affected Ka very differently. In one case, Ka was reduced substantially no matter which base was substituted for an adenine. At the other position, when a uracil was substituted with a purine, Ka decreased 10-100-fold, whereas when it was substituted by a cytosine, Ka increased about 5-fold. These studies indicate that the protein and the RNA hairpin loop interact over an extensive area and that several different types of contacts form to stabilize the complex.

Base Sequence↗
PubMed1983

Sequence-specific interaction of R17 coat protein with its ribonucleic acid binding site.

The interaction between phage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA--protein interaction. Nuclease protection and selection experiments define the binding site to about 20 contiguous nucleotides which form a hairpin. A nitrocellulose filter retention assay is used to show that the binding between the coat protein and a synthetic 21-nucleotide RNA fragment conforms to a simple bimolecular reaction. Unit stoichiometry and a Kd of about 1 nM are obtained at 2 degrees C in buffer containing 0.19 M salt. The interaction is highly sequence specific since a variety of RNAs failed to compete with the 21-nucleotide fragment for coat protein binding.

Amino Acid Sequence↗
PubMed1983

Kinetic and thermodynamic characterization of the R17 coat protein-ribonucleic acid interaction.

A filter retention assay is used to examine the kinetic and equilibrium properties of the interaction between phage R17 coat protein and its 21-nucleotide RNA binding site. The kinetics of the reaction are consistent with the equilibrium association constant and indicate a diffusion-controlled reaction. The temperature dependence of Ka gives delta H = -19 kcal/mol. This large favorable delta H is partially offset by a delta S = -30 cal mol-1 deg-1 to give a delta G = -11 kcal/mol at 2 degrees C in 0.19 M salt. The binding reaction has a pH optimum centered around pH 8.5, but pH has no effect on delta H. While the interaction is insensitive to the type of monovalent cation, the affinity decreases with the lyotropic series among monovalent anions. The ionic strength dependence of Ka reveals that ionic contacts contribute to the interaction. Most of the binding free energy, however, is a result of nonelectrostatic interactions.

Capsid↗
PubMed1983

Sequences related to HLA-DR alpha chain on human chromosome 6: restriction enzyme polymorphism detected with DC alpha chain probes.

Three sets of cosmid clones--containing the HLA-DR alpha chain gene and two additional related genes--were isolated from human genomic DNA libraries by using a cDNA probe for the HLA-DR alpha chain. Southern blot analysis using DNA from somatic cell hybrids indicated that all of the clones mapped to chromosome 6. Partial sequence analysis showed that the two additional related genes were highly homologous to each other, and to the HLA-DR alpha chain, in parts of the exon that encoded the alpha 2 domain but were more divergent in intron sequences. One of the genes corresponds to the HLA-DR-related DC series. DNA probes made from this gene revealed marked restriction enzyme polymorphism when hybridized to genomic DNA from HLA-DR typed homozygous cell lines. The patterns obtained from a number of homozygous and heterozygous cell lines correlated with the HLA-DR crossreactive serotypes and also indicated that there is a further sequence in the haploid human genome that is closely homologous with the DC alpha chain sequence. One family was studied and showed the expected HLA-DR-associated inheritance of restriction enzyme patterns. No polymorphism has yet been demonstrated in restriction enzyme fragments that include the other cloned sequence, which may correspond to the SB alpha chain gene or to a novel HLA-DR-related gene. These experiments indicate that there are at least three sequences in the human genome related, but not identical, to the HLA-DR alpha chain gene.

Base Sequence↗
PubMed1983

Interaction of R17 coat protein with its RNA binding site for translational repression.

The interaction between bacteriophage R17 coat protein and its RNA binding site for translational repression was studied as an example of a sequence-specific RNA-protein interaction. A nitrocellulose filter retention assay is used to demonstrate equimolar binding between the coat protein and a synthetic 21 nucleotide RNA fragment. The Kd at 2 degrees C in a buffer containing 0.19 M salt is about 1 nM. The relatively weak ionic strength dependence of Ka and a delta H = -19 kcal/mole indicates that most of the binding free energy is due to non-electrostatic interactions. Since a variety of RNAs failed to compete with the 21 nucleotide fragment for coat protein binding, the interaction appears highly sequence specific. We have synthesized more than 30 different variants of the binding site sequence in order to identify the portions of the RNA molecule which are important for protein binding. Out of the five single stranded residues examined, four were essential for protein binding whereas the fifth could be replaced by any nucleotide. One variant was found to bind better than the wild type sequence. Substitution of nucleotides which disrupted the secondary structure of the binding fragment resulted in very poor binding to the protein. These data indicated that there are several points of contact between the RNA and the protein and the correct hairpin secondary structure of the RNA is essential for protein binding.

Base Sequence↗