Biomedical subjects

F Hayes

Publications and source records attributed to F Hayes.

At least 37 records · Page 2Linked to original sources

PubMed1994

Topological scanning of the P1 plasmid partition site.

The parS site of the P1 plasmid promotes active partition of P1 to daughter cells when the P1 ParA and ParB proteins are provided. The structure of parS was modified by substituting portions of the sequence with synthetic oligonucleotides and testing partition activity of the resulting mutants in an in vivo assay. The boundaries of the site were defined. They enclose a 74 bp region with a central integration host factor (IHF) binding region flanked by two arms containing heptamer and hexamer ParB binding motifs. The IHF binding region was shown to be important for partition activity but could be replaced by sequences containing A tracts that induce static bends in the DNA. The properties of sites with spacer sequences of different lengths inserted at one of five different locations led to the following conclusions. (1) The spacing between the heptamer and hexamer ParB binding motifs in both arms is critical for function. (2) Optimum partition activity requires that the parS site arms are bent toward each other with specific faces of the two helices facing each other. (3) Both arms show torsional rigidity in the active complex. (4) The left arm is laterally inflexible and activity is lost when it is extended unless the right arm is similarly extended. (5) The right arm is laterally flexible so that, when it is extended by an integral number of turns of the helix, it can still align properly with a left arm of wild-type length. The results suggest that right-arm flexibility is promoted by an A + T-rich region that is essential for IHF binding and lies adjacent to the IHF binding consensus motif. Inherent flexibility of this A + T-rich region also appears to account for the residual activity of parS sites in which the IHF binding consensus has been destroyed by multiple point mutations. The results are consistent with a proposed structure in which specific alignment of the parS site arms by an IHF-promoted bend allows them to be linked by bifunctional ParB protein binding. We suggest that such a structure might be involved in the specific pairing and unpairing of daughter plasmids during partition by an isomerization reaction.

Bacterial Proteins↗

PubMed1994

The homologous operons for P1 and P7 plasmid partition are autoregulated from dissimilar operator sites.

The plasmid-partition regions of the P1 and P7 plasmid prophages in Escherichia coli are homologues which each encode two partition proteins, ParA and ParB. The equivalent P1 and P7 proteins are closely related. In each case, the proteins are encoded by an operon that is autoregulated by the ParA and ParB proteins in concert. This regulation is species-specific, as the P1 proteins are unable to repress the P7 par operon and vice versa. The homologous ParA proteins are primarily responsible for repression and bind to regions that overlap the operon promoter in both cases. The DNA-binding domain of the P7 autorepressor lies in the amino-terminal end of the P7 ParA protein. This region includes a helix-turn-helix motif that has a clear counterpart in the P1 ParA sequence. However, despite the common regulatory mechanism and the similarity of the proteins involved in repression, the promoter-operator sequences of these two operons are very different in sequence and organization. The operator is located downstream of the promoter in P1 and upstream of it in P7, and the two regions show little, if any, homology. How these differences may have arisen from a common ancestral form is discussed.

Amino Acid Sequence↗

PubMed1993

Specificity determinants of the P1 and P7 plasmid centromere analogs.

The cis-acting parS sites of P1 and P7 are similar in sequence and promote active partition of their respective plasmid prophages to daughter cells when the cognate Par proteins are supplied. Forty of the 94 relevant bases differ between the P1 and P7 parS sites, and the protein-site interactions show complete species specificity. A method was developed to predict which subset of the differing parS bases is responsible. When the four P1 bases thus identified were substituted into the P7 parS site, a complete switch to P1 specificity was observed. The P1-specific bases constitute two CG dinucleotide elements situated 66 bp apart. They lie within repeats of the TCGCCA sequence implicated in secondary contacts with the P1 ParB protein. The equivalent TC dinucleotides in the P7 site were found to be involved in P7 specificity. However, three other P7 bases can also contribute, including two in the heptamer repeats primarily responsible for ParB binding, and the P7-specific information shows some redundancy. The motifs containing the specificity dinucleotides and the primary ParB binding (heptamer) sites bear no obvious relationship of spacing or orientation to each other. For the ParB protein to contact both types of motif at the same time, the topology of the interaction must be complex.

Bacterial Proteins↗

PubMed1993

Fine-structure analysis of the P7 plasmid partition site.

The par region of bacteriophage P7 is responsible for active partition of the P7 plasmid prophage into daughter cells. The cis-acting partition site was defined precisely as a 75-bp sequence that was necessary and sufficient to promote correct segregation of an unstable vector plasmid when the two P7 partition proteins, ParA and ParB, were supplied in trans. Roughly the same region was necessary to exert partition-mediated incompatibility. The minimal site contains an integration host factor (IHF) protein binding site bracketed by regions containing heptamer repeat sequences that individually bind ParB. An additional sequence forms the left boundary of the site. Site-directed mutations in the latter sequence, as well as the IHF motif and the rightmost ParB box, blocked site function. Although the P7 site shares 55% sequence identity with its counterpart in bacteriophage P1, functional interactions between the partition sites and the Par proteins of the two plasmids were entirely species specific in vivo. The P1 sequence has similar IHF and ParB binding motifs, but the left boundary sequence differs radically and may define a point of species-specific contact with the Par proteins. No evidence was found for the existence of a functional P7 analog of the P1 parS core, a small subregion of the P1 site that, in isolation, acts as an enfeebled partition site with modified incompatibility properties.

Bacterial Proteins↗

PubMed1992

Cloning and partial sequencing of the proteinase gene complex from Lactococcus lactis subsp. lactis UC317.

The proteinase genes from Lactococcus lactis subsp. lactis UC317 were identified on a plasmid, pCI310, which is a deletion derivative of a cointegrate between pCI301, the 75 kb Lac Prt plasmid from UC317 and the 38.5 kb cryptic plasmid from that strain. The prt genes were cloned using a replacement cloning strategy whereby fragments from pCI310 were exchanged with the equivalent fragments in pNZ521, which contains the cloned proteinase genes from L. lactis subsp. lactis SK112. This generated two plasmids which encoded a cell-envelope-associated and a secreted proteinase, respectively. Specific regions of the UC317 structural prtP gene known to encode seven of the amino acids essential for substrate cleavage specificity were sequenced and compared with the known sequences of prt genes from L. lactis strains SK112, Wg2 and NCDO763. In spite of various differences that were detected in the nucleotide sequence of this region, it appears that these seven amino acids in strains UC317 and NCDO763 are identical, and represent a combination of three of the amino acids from SK112 and four from Wg2. These results indicate that the UC317 proteinase is a natural hybrid of the SK112 and Wg2 proteinases.

Amino Acid Sequence↗

PubMed1991

Molecular organization of the minimal replicon of novel, narrow-host-range, lactococcal plasmid pCI305.

Plasmid pCI305 is an 8.7-kb, narrow-host-range, cryptic plasmid originating from Lactococcus lactis subsp. lactis UC317. The nucleotide sequence of the pCI305 replication region was determined. A single open reading frame of 1158 bp was identified in the trans-active domain repB. The size of the predicted repB protein (46 kDa) is in close agreement with the size of the repB product visualized in vivo in Escherichia coli when repB was placed under control of the inducible phi T7 RNA polymerase promoter. In vivo substitution of the native repB promoter sequence with a Tn5-derived promoter sequence was demonstrated. repA, a 344-bp cis-acting region which is the probable pCI305 replication origin region, was noncoding, was AT-rich, and possessed a unique set of inverted and direct repeat sequences. No significant homology between repA or repB and other gram-positive replication regions was evident. Combined with the absence of a detectable single-stranded DNA intermediate during replication, these results indicate that the pCI305 replication region differs markedly from most gram-positive replicons examined to date. The presence on other lactococcal plasmids of replication regions related to that of pCI305 was demonstrated.

Amino Acid Sequence↗

PubMed1991

Monovalent cation-dependent reversible phosphorylation of ribosomal protein S8 in growth arrested Tetrahymena: kinetics of formation, phosphoamino acids, and phosphopeptides of mono-, and diphosphorylated derivatives of protein S8.

The kinetics of formation of mono-, and diphosphorylated derivatives of ribosomal protein S8 in Tetrahymena starving in the presence of Na+ have been determined, and the phosphoamino acids present in these derivatives have been identified. The mono-phosphorylated product, S8', contains only phosphoserine, and behaves kinetically as the precursor of the diphosphorylated product S8" which contains phosphoserine, and phosphothreonine. Tryptic digestion release a single major phosphoserine containing peptide from both S8' and S8", and a single phosphothreonine containing peptide from S8".

Amino Acid Sequence↗

PubMed1990

Integration and excision of plasmid DNA in Lactococcus lactis subsp. lactis.

The capacity of the 75-kb lactose-proteinase plasmid pCI301 from Lactococcus lactis subsp. lactis UC317 to recombine with the lactococcal chromosome was examined. Low-frequency integration of pCI301 sequences was detected following protoplast transformation of strain MG136Sm with total plasmid DNA from strain UC317. Excision of integrated sequences was subsequently observed at a low level. Excised sequences were rescued through recombination with and mobilization by the conjugative enterococcal plasmid pAMB1. Transconjugants harboring novel recombinant pCI301::pAMB1 plasmids, both pAMB1 and a pCI301 derivative, and pAMB1 only were isolated. The latter represents a class of transconjugant in which an elevated level of reintegration of pCI301 DNA in the recipient chromosome has occurred.

Chromosomes, Bacterial↗

PubMed1990

pAMbeta1-Associated Mobilization of Proteinase Plasmids from Lactococcus lactis subsp. lactis UC317 and L. lactis subsp. cremoris UC205.

A combination of plasmid curing and DNA-DNA hybridization data facilitated the identification of proteinase plasmids of 75 (pCI301) and 35 kilobases (pCI203) in the multi-plasmid-containing strains Lactococcus lactis subsp. lactis UC317 and L. lactis subsp. cremoris UC205, respectively. Both plasmids were transferred by conjugation to a plasmid-free background only after introduction of the conjugative streptococcal plasmid, pAMbeta1. All Prt transconjugants from matings involving either donor contained enlarged recombinant Prt plasmids. UC317-derived transconjugants were separable into different classes based on the presence of differently sized cointegrate plasmids and on segregation of the pCI301-derived Lac and Prt markers. All UC205-derived transconjugants harbored a single enlarged plasmid that was a cointegrate between pCI203 and pAMbeta1. The identification of prt genes on pCI301 and pCI203 derivatives was achieved by a combination of restriction enzyme and hybridization analyses.

Journal Article↗

PubMed1990

Identification of the Minimal Replicon of Lactococcus lactis subsp. lactis UC317 Plasmid pCI305.

Replication functions of the stable, cryptic 8.7-kilobase (kb) plasmid pCI305 from multi-plasmid-containing Lactococcus lactis subsp. lactis UC317 were studied. Analysis of this replicon was facilitated by the construction of replication probe vectors that consisted of the pBR322 replication region, a pUC18-derived multiple cloning site, and either the cat gene of pC194 (pCI341; 3.1 kb) or the erm gene of pAMbeta1 (pCI3330; 4.0 kb). Plasmid pCI305 was introduced into plasmid-free L. lactis subsp. lactis MG1363Sm, a streptomycin-resistant derivative of MG1363, by a transformation procedure with the 75-kb lactose-proteinase plasmid pCI301 of UC317 as a marker plasmid. A combination of transposon Tn5 mutagenesis and subcloning in pCI341 and pCI3330 with individual Tn5 insertions around the replication region facilitated the identification of a 1.6-kb minimal replicon on pCI305. This region was separable into two domains: (i) a 1.3-kb region (repB) encoding a trans-acting function (in vitro transcription-translation studies suggested the involvement of a 48-kilodalton protein); and (ii) a 0.3-kb region (repA) sufficient to direct replication when provided with repB in trans and thus probably containing the origin of replication. Lactococcus-Escherichia coli shuttle vectors based on the pCI305 replication region were constructed.

Journal Article↗

PubMed1990

High-frequency, site-specific recombination between lactococcal and pAM beta 1 plasmid DNAs.

In vivo recombination events involving the 75-kilobase lactose proteinase plasmid pCI301 of Lactococcus lactis subsp. lactis UC317 and the conjugative enterococcal plasmid pAM beta 1 were analyzed. A fragment, identified as containing the pCI301 recombination site, mediated greatly elevated levels of mobilization and recombination with pAM beta 1 when cloned in a nonmobilizable L. lactis-Escherichia coli shuttle vector. This latter recombination event was site and orientation specific on both plasmids. Recombination on pAM beta 1 was within the region associated with plasmid replication, but no effect on pAM beta 1 replication functions was detected. Resolution of recombinant plasmids generated derivatives indistinguishable from the parental plasmids.

Conjugation, Genetic↗

PubMed1989

Ribosomal proteins of Tetrahymena thermophila. Correlation of one- and two-dimensional electrophoretic migration patterns and characterization of additional small and large subunit proteins.

Further analysis of the protein complement of the cytoplasmic ribosome of the protozoon Tetrahymena thermophila has led to the identification and characterization of seven additional proteins, three in the small and four in the large subunit of this ribosome. Several of these proteins are poorly soluble or insoluble in the absence of high concentrations of urea and are not seen in the electrophoretic distribution patterns of ribosomal proteins in two-dimensional polyacrylamide gels unless 6 M urea is added to electrode buffers in contact with protein samples (first dimension) and first-dimension gels (second dimension). The migration patterns of the 40S and 60S subunits of the T. thermophila ribosome in one-dimensional polyacrylamide SDS gels and in two-dimensional gels prepared by means of the basic-acidic system of Kaltschmidt and Wittmann**, and the basic-SDS system of Zinker and Warner*** have been correlated.

Animals↗

PubMed1989

Protein-RNA crosslinking in the subunits of the cytoplasmic ribosome of Tetrahymena thermophila.

Use of 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide to introduce RNA-protein crosslinks in the 40S and 60S subunits of the cytoplasmic ribosome of Tetrahymena thermophila is described, and proteins linked covalently to 17S and 26S ribosomal RNAs are identified. RNA-protein crosslinking is accompanied by extensive dimerization and aggregation of ribosomal subunits probably due to formation of interparticle protein-protein crosslinks.

Animals↗

PubMed1988

18th Sir Hans Krebs lecture. Knowledge-based protein modelling and design.

A systematic technique for protein modelling that is applicable to the design of drugs, peptide vaccines and novel proteins is described. Our approach is knowledge-based, depending on the structures of homologous or analogous proteins and more generally on a relational data base of protein three-dimensional structures. The procedure simultaneously aligns the known tertiary structures, selects fragments from the structurally conserved regions on the basis of sequence homology, aligns these with the 'average structure' or 'framework', builds on the loops selected from homologous proteins or a wider database, substitutes sidechains and energy minimises the resultant model. Applications to modelling an homologous structure, tissue plasminogen activator on the basis of another serine proteinase, and to modelling an analogous protein, HIV viral proteinase on the basis of aspartic proteinases, are described. The converse problem of ab initio design is also addressed: this involves the selection of an amino acid sequence to give a particular tertiary structure, in this case a symmetrical domain of two Greek-key motifs.

Base Sequence↗

PubMed1987

Development of a tissue culture model for gonococcal invasion.

Neisseria gonorrhoeae invasion of the human endometrial cell line HecIB was monitored by electron microscopy. Within six hours postinfection, the gonococci have attached to the surface of some HecIB cells and are embraced by microvilli. Gonococci subsequently enter the HecIB cells in membrane bound vesicles but by eight hours, gonococci can be seen free in the cytoplasm. At twelve hours post-infection some HecIB cells are observed containing hundreds of internalized bacteria. At twenty-four hours gonococci appear in large clusters embedded in a matrix of cellular debris, which are possibly the remains of lysed infected cells. In contrast, N. lactamica is adherent to the monolayer but noninvasive.

Adenocarcinoma↗

PubMed1987

5S RNA-protein complexes released by EDTA treatment of 60S ribosomal subunits of Tetrahymena thermophila.

Treatment of large (60S) subunit of the cytoplasmic ribosome of the protozoa Tetrahymena thermophila with EDTA causes quantitative release of 5S rRNA associated with variable non quantitative amounts of one or more of 60S proteins L4, L15, L24, L31 and L41. The composition of the group of proteins released with 5S rRNA depends on both the molar ratio of EDTA and 60S subunits and the concentration of 60S subunits, in treatment mixtures.

Animals↗

PubMed1983

Ribosomal subunits and ribosomal proteins of Tetrahymena thermophila. Effect of the presence of iodoacetamide during ribosome extraction on the properties of the subunits.

Proteolytic degradation of ribosomal proteins occurs during the preparation of subunits of the cytoplasmic ribosomes of the protozoa Tetrahymena thermophila and the isolated subunits are inactive. Addition of 5 mM iodoacetamide to cell suspensions before extraction inhibits proteolytic activity and permits isolation of active subunits. The protein complements of these subunits have been characterized in two different two-dimensional electrophoretic systems, and their molecular weights have been determined.

Animals↗

PubMed1981

Properties of ribosomes and ribosomal RNAs synthesized by Escherichia coli grown in the presence of ethionine. Normal maturation of ribosomal RNA in the absence of methylation.

Analysis of 16-S rRNA synthesized in Escherichia coli D10 (met-) incubated in a medium containing ethionine in place of methionine shows that it lacks most and probably all of the methyl groups present in normal 16-SrRNA but possesses the same 3'-OH, and 5'-phosphate terminal sequences as the latter. 23-S rRNA formed in ethionine-treated cells also contains normal terminal sequences. 5-S rRNAs of normal and ethionine-treated E. coli D10 are identical. These results lead to the conclusion that methylation of ribosomal precursor RNAs is not necessary for their maturation to products with normal chain lengths and does not influence the conformation of 16-S rRNA.

Escherichia coli↗