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G M Sanders

Publications and source records attributed to G M Sanders.

29 records · Page 2Linked to original sources
PubMed1995

Rules governing the efficiency and polarity of loading a tracking clamp protein onto DNA: determinants of enhancement in bacteriophage T4 late transcription.

The bacteriophage T4 DNA polymerase accessory proteins confer processivity and high speed on replicative DNA chain elongation: the gene 45 protein, gp45, tracks along DNA and serves as the sliding clamp of the viral DNA polymerase; the gene 44/62 protein complex, gp44/62, is an ATP-dependent loading enzyme that mounts gp45 on DNA. Gp45 also activates T4 late transcription. Transcriptional enhancement by gp45 requires a particular orientation that is imposed by gp44/62 at the DNA loading site. Loading and orienting gp45 on DNA, tracking along DNA and interaction with RNA polymerase have been analyzed by measuring transcriptional activation. The efficiency of loading gp45 at different DNA structures and the resulting transcriptional activation have been compared, and sources of interference with transcriptional activation have been examined. All observations are compatible with a mechanism in which the loading enzyme recognizes the polarity of single-stranded DNA and imposes a corresponding polarity of DNA entry on gp45. Primer-template junctions are the most efficient DNA loading sites for gp45 and can generate very rapid opening at promoters that are located at a distance of > 1 kbp. In contrast, gp45 does not track efficiently across single-stranded DNA.

Bacteriophage T4↗
PubMed1995

The COOH-terminal domain of the RNA polymerase alpha subunit in transcriptional enhancement and deactivation at the bacteriophage T4 late promoter.

Many activator proteins generate their positive control of transcription through interactions with the COOH-terminal domain of the Escherichia coli RNA polymerase alpha subunit. We have examined the participation of this alpha-domain in transcriptional enhancement and suppression at bacteriophage T4 late promoters. Enhancement is generated by the T4 gene 45 protein, which is the DNA-tracking processivity factor of viral DNA replication; suppression of unenhanced transcription is generated by the RNA polymerase-binding co-activator T4 gene 33 protein. Enhanced and unenhanced transcription by RNA polymerase reconstituted with intact and truncated alpha subunits and by RNA polymerase containing ADP-ribosylated alpha has been compared; the internal structures of transcription complexes formed with these RNA polymerases have also been analyzed by footprinting and photocross-linking. Comparison of these structural and functional analyses suggests that enhancement of T4 late transcription by gp45 is not compatible with any significant role of the COOH-terminal domain of the RNA polymerase core alpha subunit in transcriptional initiation. Suppression of unenhanced T4 late transcription by the gene 33 protein also does not require the COOH-terminal domain of alpha.

Bacteriophage T4↗
PubMed1995

Old phage, new insights: two recently recognized mechanisms of transcriptional regulation in bacteriophage T4 development.

The regulation of bacteriophage T4 middle and late gene expression involves previously unrecognized mechanisms. Middle transcription requires a DNA-binding transcriptional activator and a sigma 70-binding co-activator. The coupling of late transcription to DNA replication is effected by a DNA-tracking protein that is loaded onto DNA by an assembly factor at enhancer-like entry sites. Late transcription also requires an RNA polymerase core-binding co-activator. The co-activators of T4 middle and late transcription share the property of depressing unactivated, basal transcription.

Bacteriophage T4↗
PubMed1995

Protein kinase A is a positive regulator of spore coat gene transcription in Dictyostelium.

The cotA, cotB, and cotC genes encode the major spore coat proteins of Dictyostelium. All three cot genes are coordinately expressed as aggregation is nearing completion. Induction and maintenance of their expression is dependent upon the presence of extracellular cAMP. We show that expression of a dominant inhibitor of the cAMP dependent protein kinase (PKA) in prespore cells greatly reduces the transcription rates of the cotB and cotC genes. All three cot genes contain, in their upstream regulatory regions, short sequence elements that have a high content of cytosine and adenosine residues. These CA-rich sequences are essential for optimal cot gene transcription. We show that expression of the dominant PKA inhibitor results in a greatly reduced level of the binding activity that recognizes the CA-rich sequences upstream of the cotB gene. Thus PKA acts, either directly or indirectly, to control expression of the cot genes and it may do so by modulating the activity of a DNA binding protein. However, we find that mutant cells where PKA is constitutively active still require exogenous cAMP for optimal cot gene expression in dissociated cells, suggesting that a separate, PKA-independent, signalling pathway is also involved in the regulation of cot gene expression by extracellular cAMP.

Animals↗
PubMed1994

Use of a macromolecular crowding agent to dissect interactions and define functions in transcriptional activation by a DNA-tracking protein: bacteriophage T4 gene 45 protein and late transcription.

We have used a molecular crowding reagent to define functions in the transcriptional activation of bacteriophage T4 late genes. This activation normally requires the three T4 DNA polymerase accessory proteins encoded by T4 genes 44, 62, and 45 (the gp44/62 complex and gp45), an enhancer-like cis-acting site, an RNA polymerase-bound coactivator, and an unobstructed path along the DNA joining the promoter to the enhancer. We show that molecular crowding eliminates the requirement for the gp44/62 complex and for the enhancer, retains the requirement for gp45 and its coactivator, and generates activated promoter complexes with nearly unchanged DNase I footprints. These experiments identify gp45 as the direct activator of transcription, and the gp44/62 complex as the assembly factor for gp45. They suggest that the enhancer serves as the normal, but not invariably essential, entry site for the gp45 DNA-tracking protein.

Adenosine Triphosphate↗
PubMed1994

Production of monoclonal antibodies against Xanthomonas campestris pv. mangiferaeindicae and their use to investigate differences in virulence.

Four Xanthomonas campestris pv. mangiferaeindicae isolates from mango black spot lesions were grouped according to differences in virulence and used to raise monoclonal antibodies (mAbs). Two immunization approaches were followed. In the first, four groups of mice were immunized, each with a different isolate and the spleens from each group homogenized together for cell fusion. The second approach entailed immunization of a single group of mice with bacteria pooled from all four isolates. The resultant mAbs were characterized with regard to the antigen binding specificity and antibody class. A relationship between mAb binding specificity and virulence of the bacteria was shown by Western blot analysis.

Animals↗
PubMed1992

Allergic reactions to penicillin during in vitro fertilization and intrauterine insemination.

Two cases are reported in which hypersensitivity reactions occurred after intrauterine placement of spermatozoa or embryos. Because the cells were processed and transferred in antibiotic-containing media, these reactions were suspected to be because of penicillin allergy. One patient had no prior penicillin allergy but was found to be penicillin allergic by skin testing. The other had a history of allergy to oral penicillin. In both cases, the allergy symptoms did not occur during subsequent cycles when antibiotics were removed from the transfer media. These reports suggest that in patients known to be penicillin sensitive, penicillin should not be used during transfer of gametes and embryos for assisted reproductive procedures. In addition, the routine use of antibiotics in these procedures should be scientifically evaluated.

Adult↗