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Biomedical subjects

A Merryweather

Publications and source records attributed to A Merryweather.

10 recordsLinked to original sources
PubMed2000

Membrane type 4 matrix metalloproteinase (MMP17) has tumor necrosis factor-alpha convertase activity but does not activate pro-MMP2.

Membrane type 4 matrix metalloproteinase (MT4-MMP) shows the least sequence homology to the other MT-MMPs, suggesting a distinct function for this protein. We have isolated a complete cDNA corresponding to the mouse homologue which includes the signal peptide and a complete pro-domain, features that were lacking from the human form originally isolated. Mouse MT4-MMP (mMT4-MMP) expressed in COS-7 cells is located at the cell surface but does not show ability to activate pro-MMP2. The pro-catalytic domain was expressed in Escherichia coli as insoluble inclusions and active enzyme recovered after refolding. Activity of the isolated catalytic domain against synthetic peptides commonly used for MMP enzyme assays could be inhibited by TIMP1, -2, and -3. The recombinant mMT4-MMP catalytic domain was also unable to activate pro-MMP2 and was very poor at hydrolyzing components of the extracellular matrix with the exception of fibrinogen and fibrin. mMT4-MMP was able to hydrolyze efficiently a peptide consisting of the pro-tumor necrosis factor alpha (TNFalpha) cleavage site, a glutathione S-transferase-pro-TNFalpha fusion protein, and was found to shed pro-TNFalpha when co-transfected in COS-7 cells. MT4-MMP was detected by Western blot in monocyte/macrophage cell lines which in combination with its fibrinolytic and TNFalpha-converting activity suggests a role in inflammation.

ADAM Proteins↗
PubMed1997

Plant-derived vaccine protects target animals against a viral disease.

The successful expression of animal or human virus epitopes on the surface of plant viruses has recently been demonstrated. These chimeric virus particles (CVPs) could represent a cost-effective and safe alternative to conventional animal cell-based vaccines. We report the insertion of oligonucleotides coding for a short linear epitope from the VP2 capsid protein of mink enteritis virus (MEV) into an infectious cDNA clone of cowpea mosaic virus and the successful expression of the epitope on the surface of CVPs when propagated in the black-eyed bean, Vigna unguiculata. The efficacy of the CVPs was established by the demonstration that one subcutaneous injection of 1 mg of the CVPs in mink conferred protection against clinical disease and virtually abolished shedding of virus after challenge with virulent MEV, demonstrating the potential utility of plant CVPs as the basis for vaccine development. The epitope used occurs in three different virus species-MEV, canine parvovirus, and feline panleukopenia virus- and thus the same vaccine could be used in three economically important viral hosts-mink, dogs, and cats, respectively.

Amino Acid Sequence↗
PubMed1996

Cloning and initial characterization of 14 myb-related cDNAs from tomato (Lycopersicon esculentum cv. Ailsa Craig).

myb-related transcription factors contain highly conserved DNA-binding domains. Using a mixture of degenerate oligonucleotides derived from the highly conserved region as probe, 14 myb-related clones were isolated from a cDNA library constructed using tomato hypocoyl mRNA. The expression of these clones was studied by northern blot analysis using poly(A)+ RNA from 7 tissue types (hypocotyl, leaf, root, green and red fruit, immature and mature flower). This study has revealed a wide range of expression patterns which include multiple and single transcripts, some of which show marked tissue specificity. Two clones showing different expression patterns have been fully sequenced. The DNA-binding domains of these two tomato myb clones are compared with myb genes from other plant species and organisms. Of the three clones analysed so far by Southern hybridization, two are single-copy genes and one has multiple genomic copies.

Amino Acid Sequence↗
PubMed1992

Control of gene expression in tobacco cells using a bacterial operator-repressor system.

We have investigated the efficacy of using the Escherichia coli lac operator-repressor system to control plant gene expression. The lacI gene was modified to allow optimal expression in plant cells and then placed downstream of the cauliflower mosaic virus (CaMV) 35S RNA promoter. This construct was introduced into tobacco plants by leaf disc transformation. Transgenic tobacco plants synthesized significant quantities of LacI protein (up to 0.06% of total soluble protein). We have used the E.coli beta-glucuronidase gene (gus) as the reporter gene by placing it downstream of the maize chlorophyll a/b binding protein (CAB) gene promoter. Lac operators were introduced into several positions within the CAB promoter and operator-free plasmid was used as control. Repression was assessed by comparing the transient expression from CAB-operator-gus reporter constructs in protoplasts expressing lac protein, with that in control cells not expressing the repressor. Repression varied between 10 and 90% with different operator positions. Transient assays were also performed in the presence of the inducer, isopropyl-beta-D-thiogalactoside (IPTG). In lacI protoplasts the presence of IPTG manifested itself in a 4.2-fold relief of repression. The study was extended to show regulation of expression in stable transformants. Tobacco transformants harbouring a CAB-operator-gus reporter construct and the lacI gene were shown to have repressed GUS levels, but in the presence of IPTG, repression was relieved 15-fold. We conclude that the lac repressor can enter the plant cell nucleus, find its cognate operator sequence in the chromatin to form a repressor--operator complex and effectively block transcription of a downstream gene.

Bacterial Proteins↗
PubMed1992

Efficient repression by a heterodimeric repressor in Escherichia coli.

Previous studies with purified variants of the 434 repressor having different operator-binding specificities have demonstrated the interactions of a heterodimeric repressor with a hybrid operator site. The present study investigates the interactions between the 434 repressor and its operator site. The optimum 434 operator half-site is used with a P22 operator half-site to create a hybrid 434/P22 operator. We show that this hybrid operator can be efficiently bound by a heterodimeric repressor, consisting of one wild-type 434 repressor monomer and one 434 repressor monomer with the binding specificity of the P22 repressor, to bring about repression in Escherichia coli.

Amino Acid Sequence↗
PubMed1989

Overinitiation of replication of the Escherichia coli chromosome from an integrated runaway-replication derivative of plasmid R1.

A 16-base-pair fragment, deletion of which completely inactivated oriC, was replaced by a temperature-dependent runaway-replication derivative (the copy number of which increases with temperature) of the IncFII plasmid R1. The constructed strains were temperature sensitive, and flow cytometry revealed a severalfold increase in the DNA/mass ratio following shifts to nonpermissive temperatures. The cell size distribution was broader in the constructed strains relative to that in the wild type because of asynchrony between the chromosome replication and cell division cycles. This difference was more pronounced for counterclockwise initiation of chromosomal replication, in which small DNA-less cells and long filaments were abundant. Following a temperature shift the cell size distributions became even more broad, showing that changes in the frequency of chromosomal replication affect cell division and emphasizing the interplay between these two processes.

Chromosomes, Bacterial↗
PubMed1987

Direct selection for the exchange of alleles between a plasmid and the Escherichia coli chromosome.

Recombination is extensively used in order to move alleles between replicons. The exchange of wild-type chromosomal and mutant plasmid-borne alleles is a two-step process entailing the formation of a cointegrate between the entire plasmid and the chromosome, followed by resolution of such cointegrates to give a mutant chromosome and a plasmid carrying the wild-type chromosomal sequence. Often the cointegrate and the resolved forms cannot be distinguished phenotypically. To enable the direct isolation of the resolved products we have developed a positive selection technique. Cells containing a cointegrated plasmid R1 were constructed by transduction using a P1 lysate prepared from cells harbouring a plasmid comprising a mutant chromosomal allele and the so-called omega fragment which carries an aad (aminoglycoside adenylyltransferase) gene. P1 transduction from the cointegrate strain into an SmD recipient allowed direct selection for the resolved complex, since transduction of the aad gene is lethal to an SmD strain.

Alleles↗
PubMed1986

Role of sog polypeptides specified by plasmid ColIb-P9 and their transfer between conjugating bacteria.

The sog gene of the conjugative plasmid ColIb-P9 specifies two sequence-related polypeptides with the N-terminal third of the larger product having DNA primase activity. To resolve the function of the C-terminal portion of the polypeptides, we constructed a ColIb mutant containing a Tn5 insertion in the 3' region of sog. The mutation truncated sog gene products without inactivating DNA primase and rendered the plasmid defective in conjugation. Tests for the presence of conjugative pili, for complementation by a sog+ recombinant, and for mobilization of small origin of transfer (oriT) recombinant plasmids indicated that the mutant ColIb allows conjugative aggregation of cells but it is defective in DNA transfer at some stage subsequent to its initiation at oriT. Physical evidence is given that normal sog polypeptides are among a group of proteins transferred selectively from the donor to the recipient cell by a conjugation-specific process. No transfer of the mutant sog proteins was detected. It is proposed that the C-terminal region of sog polypeptides facilitates transfer of single-stranded ColIb DNA between conjugating cells following initiation of transfer at the oriT site, and that in this role the proteins are transmitted to the recipient cell.

Bacterial Proteins↗
PubMed1986

Role and specificity of plasmid RP4-encoded DNA primase in bacterial conjugation.

The role of the DNA primase of IncP plasmids was examined with a derivative of RP4 containing Tn7 in the primase gene (pri). The mutant was defective in mediating bacterial conjugation, with the deficiency varying according to the bacterial strains used as donors and recipients. Complementation tests involving recombinant plasmids carrying cloned fragments of RP4 indicated that the primase acts to promote some event in the recipient cell after DNA transfer and that this requirement can be satisfied by plasmid primase made in the donor cell. It is proposed that the enzyme or its products or both are transmitted to the recipient cell during conjugation, and the role of the enzyme in the conjugative processing of RP4 is discussed. Specificity of plasmid primases was assessed with derivatives of RP4 and the IncI1 plasmid ColIb-P9, which is known to encode a DNA primase active in conjugation. When supplied in the donor cell, neither of the primases encoded by these plasmids substituted effectively in the nonhomologous conjugation system. Since ColIb primase provided in the recipient cell acted weakly on transferred RP4 DNA, it is suggested that the specificity of these enzymes reflects their inability to be transmitted via the conjugation apparatus of the nonhomologous plasmid.

Conjugation, Genetic↗