PubMed Health⌕ Search

Biomedical subjects

C Hadfield

Publications and source records attributed to C Hadfield.

13 recordsLinked to original sources
PubMed1995

Protein binding interactions at the STB locus of the yeast 2 microns plasmid.

The cis-acting STB locus has been shown to be a multiple protein binding site. STB-specific binding activity was detected in a normally insoluble yeast cell protein fraction, suggesting association with a subcellular structure. Both 2 microns-encoded and host-encoded STB-binding activities were identified. The 2 microns proteins showed contrasting STB-binding activities: C (REP2) protein acted cooperatively with the host factor to promote STB binding; B (REP1) protein also acted in association with the host factor, but showed a dual action, opposing or facilitating binding, depending upon concentration; D (RAF) exhibited rapid binding and antagonism to host factor binding. FLP did not bind, but promoted host factor dissociation. The implications of these activities for the molecular mechanism of 2 microns plasmid inheritance are considered.

Base Sequence↗
PubMed1995

Determination of chromosome copy numbers in Saccharomyces cerevisiae strains via integrative probe and blot hybridization techniques.

Methods have been devised for analyzing chromosome copy numbers in S. cerevisiae strains that may be polyploid or aneuploid, as is apparent in the case of many industrial strains. The initial step involved transformation of a strain with an integrative "ploidy probe" transplacement fragment that enabled the copy number of the targeted chromosomal locus to be determined via genomic Southern blotting and quantitative probe hybridization. Dual probe co-hybridization to Southern genomic DNA blots was used to extend such locus copy number determinations to other loci within the same chromosome, thereby screening for internal consistency along the length of the chromosome. This approach was also used to extend the analysis to other chromosomes in the genome. The method was established and verified with euploid series laboratory strains and then used to examine chromosome copy numbers in three industrial strains. One brewing strain apparently contained three copies of the chromosomes tested, whilst another brewing and a baking strain showed evidence of aneuploidy.

Blotting, Southern↗
PubMed1990

G418-resistance as a dominant marker and reporter for gene expression in Saccharomyces cerevisiae.

Coding sequence cartridges for aminoglycoside phosphotransferase (APT) were isolated from bacterial transposon Tn903. When incorporated into a heterologous gene construction utilising the PGK1 promoter and terminator, the heterologous APT gene provided a G418-resistance determinant that functioned efficiently as a dominant marker for yeast in both multiple- and single-copy. Transformant colonies on selective medium appeared rapidly, within 36-48 h, and growth rate of the transformed cells was normal. A simple and highly sensitive radiolabelling assay for APT enzyme activity was developed for use with crude cell protein extracts. Enzyme activity units were equated to the amount of APT protein present in the cells, and the APT protein was shown to be stable in yeast. Heterologous APT expression was 130-fold reduced compared with homologous PGK1. This resulted from an estimated two-fold decrease in mRNA level and a 65-fold decrease in translation efficiency. The latter was unaffected by AUG sequence context change, but corresponded with a high frequency of minor codons in the APT-coding sequence. APT can be used as a semi-quantitative reporter of gene expression, whose useful features are in vivo detection via the G418-resistance phenotype and powerful cell-free assay.

Amino Acid Sequence↗
PubMed1987

Sequence and expression characteristics of a shuttle chloramphenicol-resistance marker for Saccharomyces cerevisiae and Escherichia coli.

An efficiently transforming chloramphenicol-resistance (CmR) shuttle marker for Saccharomyces cerevisiae and Escherichia coli has been characterized in terms of its primary structure and expression characteristics. The complete nucleotide (nt) sequence of the CmR marker is given, with details on restriction sites, apparent expression signals for both organisms, and translation of the Cm acetyltransferase (CAT)-coding sequence. SDS-polyacrylamide gel electrophoresis and Western blotting have confirmed that the marker produced an identical CAT protein in yeast and E. coli. Each copy of the marker, whether present in multiple copies or as a single copy, gave rise to approx. 0.1% of the total soluble protein as CAT in haploid yeast cells. When compared with homologous expression of alcohol dehydrogenase (ADH-I) by the same ADC1 promoter, this represents a 27-fold reduction for CAT expression, which is typical of heterologous gene expression in yeast. When the marker was on a multicopy plasmid in yeast, up to 2.1% of the total soluble cell protein was produced as CAT, but this did not adversely affect the growth of host cells. Increase of the Cm concentration in the medium did not result in an increase in the number of plasmids nor the amount of CAT protein produced, showing that plasmid copy number and marker expression are regulated independently of the selection pressure. In E. coli, the ADC1 yeast-promoter DNA was found to contain both forwards and backwards promoter activity. The level of expression provided by these promoters was equivalent to that of an average E. coli gene.

Acetyltransferases↗
PubMed1986

An efficient chloramphenicol-resistance marker for Saccharomyces cerevisiae and Escherichia coli.

Chloramphenicol (Cm) was demonstrated to be a suitable selective agent for the plasmid-mediated transformation of haploid and polyploid strains of Saccharomyces cerevisiae. A yeast/Escherichia coli shuttle Cm-resistance (CmR) marker was constructed by inserting the CAT coding sequence from Tn9, and its associated bacterial ribosome-binding site, between a modified yeast ADC1 promoter and CYC1 terminator. When present on a 2 microns-based replicating plasmid, this marker transformed yeast as efficiently as the auxotrophic markers TRP1 and LEU2. When included in an integrating vector, single-copy transformants were formed as efficiently as with LEU2 and HIS3. Industrial yeast strains were transformed with both the replicating and integrating plasmids. The CmR marker could also efficiently transform E. coli. This versatile and efficient performance is currently unique for a yeast dominant marker.

Acetyltransferases↗
PubMed1984

Value of urine microscopy in predicting histological changes in the kidney: double blind comparison.

Fresh, first morning specimens of urine from 22 consecutive patients were examined by quantitative microscopy on the morning of renal biopsy; the renal biopsy samples were evaluated "blindly." Five patients showed no abnormality in the biopsy samples but eight had minimal, one mild, six moderate, and two severe histological changes. Comparison of the results of quantitative microscopy of urine with the presence or absence of histological evidence of disease showed that sensitivity was 88%, specificity 83%, accuracy 86%, positive predictive value 93%, and negative predictive value 71%. When combined with microscopy of a second urine specimen these values were 100%, 50%, 87%, 85%, and 100% respectively. There was a significant relation between number of casts and severity of the histological changes (p less than 0.01). Comparison of renal functional abnormalities with histological findings gave values of 64%, 100%, 73%, 100%, and 50% respectively. It is concluded that quantitative microscopy of the first morning specimen of urine is a sensitive test with high predictive value for the presence or absence of renal disease. If no casts are detected in two early morning specimens the likelihood of finding anything more than minimal changes in a biopsy sample is virtually zero.

Biopsy↗
PubMed1984

Molecular analysis of the period locus in Drosophila melanogaster and identification of a transcript involved in biological rhythms.

We have isolated and analyzed DNA sequences encompassing the period (per) locus of Drosophila melanogaster. The location of this clock gene was delimited by the molecular mapping of chromosome aberrations at or very near the per locus. At least five RNAs are transcribed from this region. One of these transcripts, a 0.9 kb species, is strongly implicated in per's control of biological rhythms. Two independently isolated arrhythmic mutations at the per locus dramatically reduce the level of this transcript. Furthermore, the level of the 0.9 kb transcript is strongly modulated during a light/dark cycle. We discuss evidence, from previously reported genetic and phenotypic analysis of per's function, suggesting that this region may be complex and that several gene products from the per region, including this 0.9 kb transcript, may be involved in the different aspects of normal rhythmicity influenced by this clock gene.

Animals↗
PubMed1984

A genetic analysis of defective plasmid formation by N-deficient phage lambda chromosomes.

Plasmid formation by N- derivatives of lambdoid phages has been reinvestigated with transducing phages carrying the trp, lac and gal genes of Escherichia coli. Transduction by lambda N- cI- derivatives was inefficient and short-lived in each case, under both selective and nonselective conditions. Mutant-operators were introduced to relieve possible auto-repression by the cro gene product. Such N-defective phage genomes were able to propagate continually as plasmids, although without selection they were gradually lost from the carrier cells. Plasmid formation remained inefficient, however. The entire chromosome of N- phages can be expressed by transcription that leaks through the serially arranged Rho-dependent terminators. Some functions so expressed are deleterious to the plasmid state and cause the instability of lambda N- plasmids.

Bacteriophage lambda↗
PubMed1984

Functions antagonistic to plasmid formation by lambda N- chromosomes.

It was concluded in the preceding paper that lambda N- cI- genomes probably failed to form plasmids within infected Escherichia coli cells because they leakily express functions that act to destabilize the plasmid state. This prediction was investigated by examining the effect upon plasmid-forming ability of the loss of possible anti-plasmid functions. The loss of Ter function was found to allow long-term plasmid formation, although the efficiency of initial plasmid formation and the heritable stability without selection were low. The combined loss of the int, red and gam gene functions also promoted plasmid growth, although the absence of Ter lambda was necessary. In contrast, the presence of Ter80 (due to an h80 substitution) did not prevent plasmid formation when the int, red and gam genes were absent, indicating that Ter80 does not attack the closed-circular form of the lambda chromosome. The combined loss of the ter, int, red and gam gene functions facilitated fully efficient inheritance of the lambda N- cI- plasmids in the absence of selection, although the efficiency of initial plasmid formation remained low. However, cells harbouring such plasmids suffered a decline in viability, indicating that the plasmids expressed a function (or more than one) that acts to debilitate the host cells--presumably an effect that is increased with this genotype because the modified lambda N- cI- plasmids are inherently more stable. The possible involvement of the lambda S and kil functions in destabilization is discussed.

Autoradiography↗
PubMed1984

A programme for the construction of a lambda phage.

Infection of a sensitive host by the lambdoid coliphages can cause death of the bacterial cell by lysis or can lead to a lysogenic cell, with the viral DNA stably integrated into the host chromosome. These alternative responses both require the coordination of several host and phage functions, and lambda infection follows a well controlled developmental plan. The lytic and lysogenic pathways of lambdoid infection are reviewed, with emphasis on the variety of control mechanisms involved in the commitment to a particular pathway.

Bacteriolysis↗
PubMed1979

The improving hematologic picture in long-term surviving calves with total artificial hearts.

A coagulopathy associated with severe hemolysis was a limiting factor in obtaining long-term survivors among calves with total artificial hearts in 1969. Conversion of design from sac-type hearts to flexible diaphragm hearts, and from Silastic Dacron-fibril intimas to smooth polyurethane intimas, resulted in an abatement of the coagulopathy. In the most recent series of animals studied at this laboratory, platelet counts are normal and platelet survivals are half of normal. Plasma hemoglobins are normal. The coagulation system is still activated at specific loci within the total artificial heart, but is best compensated for in calves treated with antiplatelet drugs, having polyurethane hearts.

Animals↗