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Biomedical subjects

A Greener

Publications and source records attributed to A Greener.

16 recordsLinked to original sources
PubMed1997

Using Schizosaccharomyces pombe as a host for expression and purification of eukaryotic proteins.

We have established a eukaryotic protein expression and purification system by using the yeast Schizosaccharomyces pombe as the host and the glutathione S-transferase (GST) as a protein purification tag. This system provides opportunities for rapid, inexpensive, and high yield production of proteins in a eukaryotic organism. Unlike E. coli, S. pombe provides for post-translational modifications of the proteins, which are often critical for the structure and function of eukaryotic proteins. Two vectors have been constructed for protein expression in S. pombe, pESP-1 and pESP-2. Both vectors use the nmt1 promoter for constitutive or induced expression of the gene of interest. Expressed GST-tagged proteins are easily and rapidly purified using glutathione agarose beads. The GST tag can be removed from the fusion proteins by treatment with either the thrombin or enterokinase protease. Proteins expressed from the pESP-2 vector will yield native amino acid sequence when the GST tag is removed by treatment with enterokinase. Nine proteins have been purified by using the system with yields ranging from 1.0 mg/l to 12.5 mg/l of induced culture.

Amino Acid Sequence↗
PubMed1997

An efficient random mutagenesis technique using an E. coli mutator strain.

Random mutagenesis of a cloned gene remains a central method to understand many aspects of the gene products function and structure. Having the ability to introduce a limited number of changes within a gene in a controlled fashion allows one to evaluate single changes and study the effect these variants have on the gene of interest. The in vivo random mutagenesis strategy described in this article, using an E. coli host, is a convenient method to introduce a limited number of mutations in a controlled manner.

Escherichia coli↗
PubMed1994

A Clarkson's sector integration routine for personal computers.

A Clarkson's sector integration scheme has been implemented on a personal computer to calculate mean scatter-air ratio (SAR) and mean tissue-air ratio (TAR) for external photon beams of irregular shape. To find where a given radial line from the point of interest intersects the edge of the radiation field, polygon clipping algorithms have been adapted from the field of image processing. The program has calculation run times of approximately 1 s on an 80387 with a math coprocessor for an integration of 36 sectors. The software was validated by comparison to program IRREG, where for various squares and clinical shapes, the agreement was typically 0.7%. The source code is available from the authors.

Algorithms↗
PubMed1992

Promoters of the broad host range plasmid RK2: analysis of transcription (initiation) in five species of gram-negative bacteria.

A broad host range cloning vector was constructed, suitable for monitoring promoter activity in diverse Gram-negative bacteria. This vector, derived from plasmid RSF1010, utilized the firefly luciferase gene as the reporter, since the assay for its bioluminescent product is sensitive, and measurements can be made without background from the host. Twelve DNA fragments with promoter activity were obtained from broad host range plasmid RK2 and inserted into the RSF1010 derived vector. The relative luciferase activities were determined for these fragments in five species of Gram-negative bacteria. In addition, four promoters were analyzed by primer extension to locate transcriptional start sites in each host. The results show that several of the promoters vary substantially in relative strengths or utilize different transcriptional start sites in different bacteria. Other promoters exhibited similar activities and identical start sites in the five hosts examined.

Base Sequence↗
PubMed1992

Identification of a novel promoter in the replication control region of plasmid R6K.

A novel source of transcription has been detected in the replication region of plasmid R6K by using fusions involving the galK reporter gene. The -35 and -10 consensus RNA polymerase binding sites were identified in the region overlapping the binding sites for the R6K-encoded replication protein pi. Transcription from this promoter, designated P2, is repressed in vivo by pi-protein levels that are inhibitory for replication. Promoter-down mutations in P2 induced in vitro by bisulfite mutagenesis result in a reduced copy number of a beta-replicon but not of a gamma-replicon. Implications of the role of P2 in R6K replication are discussed.

Bacterial Proteins↗
PubMed1990

N-terminal truncated forms of the bifunctional pi initiation protein express negative activity on plasmid R6K replication.

The replication initiation protein pi of the Escherichia coli plasmid R6K is a dual regulator in the control of plasmid copy number, functioning both as a specific initiator and inhibitor of replication. While the biochemical basis of these activities is not known, initiator activity requires binding of the protein to the seven 22 bp direct repeats within the gamma-origin region. By deleting C-terminal segments of the pi coding region, we have found that the N-terminal polypeptides of pi that are produced, corresponding to the first 117 and 164 amino acids, respectively, retain the negative activity of the bifunctional protein, i.e. these truncated pi proteins specifically inhibit R6K replication in vivo. These negatively acting polypeptides, however, are incapable of initiating replication in vivo and fail to bind to the gamma-origin of the R6K DNA in vitro. A correspondence between the observed negative activity of the N-terminal peptide and the negative regulatory activity of the intact pi protein is supported by the finding that point mutations introduced into the 164 amino acid N-terminal peptide that result in a decrease in its inhibitory activity also produce a plasmid high-copy phenotype when these mutations are incorporated into the full-length pi protein. These findings demonstrate that the negative domain of pi resides in the N-terminal segment of the protein. Furthermore, the data obtained suggest that inhibition of R6K replication by pi does not require direct binding to DNA.

Amino Acid Sequence↗
PubMed1990

Identification of human antibody fragment clones specific for tetanus toxoid in a bacteriophage lambda immunoexpression library.

We have applied a molecular biology approach to the identification of human monoclonal antibodies. Human peripheral blood lymphocyte mRNA was converted to cDNA and a select subset was amplified by the polymerase chain reaction. These products, containing coding sequences for numerous immunoglobulin heavy- and kappa light-chain variable and constant region domains, were inserted into modified bacteriophage lambda expression vectors and introduced into Escherichia coli by infection to yield a combinatorial immunoexpression library. Clones with binding activity to tetanus toxoid were identified by filter hybridization with radiolabeled antigen and appeared at a frequency of 0.2% in the library. These human antigen binding fragments, consisting of a heavy-chain fragment covalently linked to a light chain, displayed high affinity of binding to tetanus toxoid with equilibrium constants in the nanomolar range but did not cross-react with other proteins tested. We estimate that this human immunoexpression library contains 20,000 clones with high affinity and specificity to our chosen antigen.

Amino Acid Sequence↗
PubMed1987

DNA and protein interactions in the regulation of plasmid replication.

As for bacterial and animal viruses that employ different mechanisms for their duplication in a host cell, plasmids have evolved different strategies to assure their hereditary stability or maintenance at a specific copy number during cell growth and division. A characteristic feature of plasmid replication control, however, is an involvement of one or more negatively controlling elements. Furthermore, a majority of the bacterial plasmids examined to date contain direct nucleotide sequence repeats at their origin of replication and encode a replication protein that binds to these repeat sequences. The binding of the replication protein (pi protein) specified by the antibiotic resistance plasmid R6K to a set of 22 base pair direct nucleotide sequence repeats is required for the initiation of replication at each of three origins of replication (alpha, beta and gamma) within a 4 Kb segment of R6K. The pi initiation protein is multifunctional in that it has both positive and negative activities in both controlling the initiation of replication and autoregulating its own synthesis. Similarly, the direct repeats of plasmid R6K and several other plasmid systems play more than one role in plasmid replication. These repeats, termed iterons, are not only required for origin activity but also exert a negative effect on plasmid copy number possibly as a result of their 'titration' of a plasmid encoded replication protein. The properties of plasmid replication proteins and direct nucleotide sequence repeats that are important for their opposing positive and negative roles in the regulation of the initiation of replication are described with particular emphasis on plasmid R6K of Escherichia coli.

Bacterial Proteins↗
PubMed1985

Autorepressor properties of the pi-initiation protein encoded by plasmid R6K.

A DNA fusion containing the promoter of the pir gene of plasmid R6K that encodes for the pi-initiation protein and the beta-galactosidase gene of Escherichia coli (lacZ) is described. The synthesis of beta-galactosidase promoted by this pir-lac fusion was almost completely inhibited when an R6K sequence containing the pir gene was provided in trans in E. coli. Transcription in vitro from the pir promoter but not the trp promoter of E. coli, was inhibited by purified pi protein indicating that the pi protein alone is responsible for repression of its own gene and that the effect is promoter specific. The DNA-protein interaction sites in the pir regulatory region have been determined for the pi protein and E. coli RNA polymerase using the DNase I protection method. The binding sites for these two proteins overlap for three helical turns. Competition DNA binding experiments show that the pi protein will displace bound RNA polymerase. From these studies we conclude that repression of the pir gene is accomplished by binding of the pi protein and this association blocks access of RNA polymerase to the pir promoter region.

Base Sequence↗
PubMed1985

Excision and reintegration of the Escherichia coli K-12 chromosomal element e14.

The genetic element e14 is a natural component of the Escherichia coli K-12 chromosome. On induction of the SOS pathways, e14 excises as a 14.4-kilobase circle. We report here on the reintegration of e14 into the chromosome of cured (e14 degrees) E. coli K-12 derivatives. Using a Tn10 insertion mutant of e14, we found that reintegration occurred specifically at the locus originally occupied by e14 and with the same orientation. The reintegration event required neither the RecA nor the RecB functions. The attachment site of the free form was located within a 950-base-pair HindIII-AvaI fragment and shared sufficient homology with the host attachment site to form detectable DNA-DNA hybrids. Even though E. coli C and B/5 did not contain e14, they did possess a HindIII restriction fragment that hybridized to the free e14 attachment fragment. E. coli C could be transformed with e14-1272::Tn10, resulting in integration at this site of homology. The Tn10 mutants were also used in mapping the point of e14 attachment. We found the following sequence: fabD purB atte14 umuC. Furthermore, analysis of a recombinant plasmid that contained both the e14 attachment site and the purB locus showed that these two loci occur within 11 kilobases of each other.

Chromosome Mapping↗
PubMed1980

Identification of a novel genetic element in Escherichia coli K-12.

Induction of the SOS repair processes of Escherichia coli K-12 caused a 14.4-kilobase species of circular deoxyribonucleic acid, called element e14, to be excised from the chromosome. To aid further characterization of this species, an 11.6-kilobase segment of e14 was inserted into the HindIII site of plasmid pBR313. To map e14 on the E. coli K-12 chromosome, the recombinant plasmid, pAG2, was used to transform a polA recipient, an event which required integration of pAG2 into the recipient chromosome. This recombinational event was dependent upon the region of homology between the incoming plasmid and the chromosome, as no transformants were scored when either a strain cured of the element was the recipient or pBR313 was the transforming deoxyribonucleic acid. Using these transformants, we have shown that e14 maps between the purB and pyrC loci near min 25. Several strains of E. coli K-12 were found to contain e14; however, one strain, Ymel trpA36, did not. In addition, e14 was found to be absent in both E. coli B/5 and E. coli C. The approach to mapping developed for this work could be used to map other fragments of E. coli deoxyribonucleic acid which have no known phenotype.

Chromosome Mapping↗
PubMed1977

Effect of the petite mutation on maltose and alpha-methylgucoside fermentation inSaccharomyces cerevisiae.

Several hundred petite mutants were isolated from yeast strains of different genotype to examine the effect of the petite mutation on maltose and alpha-methylglucoside fermentation. In most cases petite mutants isolated retain the ability to ferment maltose and alpha-methylglucoside, although at a slower rate. In one strain (1403-7A), however, the ability to ferment alpha-methylglucoside is completely lost in all petite mutants isolated from this strain. It is suggested that mitochondrial factors may be involved in the utilization of alpha-methylglucoside in strain 1403-7A.

Erythromycin↗