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Biomedical subjects

J L Campbell

Publications and source records attributed to J L Campbell.

At least 19 recordsLinked to original sources
PubMed1992

Regulation of the yeast DNA replication genes through the Mlu I cell cycle box is dependent on SWI6.

In Saccharomyces cerevisiae, at least 17 DNA replication genes are coordinately expressed at the G1/S boundary during the cell cycle. All of these genes have the DNA sequence element ACGCGT in their 5' upstream regulatory regions. This sequence has been shown to be essential for periodic expression of the POL1, CDC9, and TMP1 genes. The cyclin (CLN1 and CLN2) and HO genes are another subset of genes that are expressed with the same timing as the DNA replication genes. Their periodic expression requires the participation of two well-characterized transcriptional activators: the SWI4 and SWI6 gene products. In this study, we present evidence that SWI6 contributes to the regulation of DNA replication genes as well. Surprisingly, a preferential requirement for SWI6 over SWI4 is observed in our studies of ACGCGT-dependent reporter gene expression in vivo. This selectivity has not been observed for the other G1/S genes. Correlating with the in vivo results, protein-DNA complexes formed in vitro on multimeric ACGCGT elements are either abolished or reduced in swi6 delta deletion mutants.

Base Sequence↗
PubMed1992

Protein stitchery: design of a protein for selective binding to a specific DNA sequence.

We present a general strategy for designing proteins to recognize DNA sequences and illustrate this with an example based on the "Y-shaped scissors grip" model for leucine-zipper gene-regulatory proteins. The designed protein is formed from two copies, in tandem, of the basic (DNA binding) region of v-Jun. These copies are coupled through a tripeptide to yield a "dimer" expected to recognize the sequence TCATCGATGA (the v-Jun-v-Jun homodimer recognizes ATGACTCAT). We synthesized the protein and oligonucleotides containing the proposed binding sites and used gel-retardation assays and DNase I footprinting to establish that the dimer binds specifically to the DNA sequence TCATCGATGA but does not bind to the wild-type DNA sequences, nor to oligonucleotides in which the recognition half-site is modified by single-base changes. These results also provide strong support for the Y-shaped scissors grip model for binding of leucine-zipper proteins.

Amino Acid Sequence↗
PubMed1992

The 66 kDa component of yeast SFI, stimulatory factor I, is hsp60.

DNA polymerase epsilon stimulatory factor I (SFI) has been shown to contain three peptides, p66, p37 and p13. Two of these components have been identified. The p66 gene was cloned by using a p66 antibody to screen a lambda gt11 library. A portion of the gene was sequenced and confirmed to encode p66 by the presence of protein sequence corresponding to that of p66 tryptic peptides. The gene was identified as HSP60 by a homology search of GenBank. Tryptic peptides of p37 were sequenced and identified as belonging to yeast translation initiation factor 4A by a homology search of PIR. The HSP60 gene maps to chromosome XII.

Amino Acid Sequence↗
PubMed1992

Identification and purification of DBF-A, a double-stranded DNA-binding protein from Saccharomyces cerevisiae.

Using oligonucleotide affinity chromatography with DNase I footprinting as an assay we have looked for proteins that interact with sequence elements within the yeast origin of replication, autonomously replicating sequence 1 (ARS1). In this work we describe a protein that binds with high affinity to DNA but displays only moderate sequence specificity. It is eluted at 0.7 M salt from an ARS1 oligonucleotide column. Footprinting analysis on ARS1 at a high protein concentration revealed at least three sites of protection flanking element A and its repeats. Element A itself is rendered hypersensitive to DNase I digestion upon protein binding. This pattern is also observed for the H4 and HMR-E ARSs, suggesting that the protein alters the DNA conformation at element A and its repeats. The affinity-purified fraction is also capable of supercoiling a relaxed, covalently closed plasmid in the presence of topoisomerase. Highly purified preparations of the protein are enriched in an 18-kDa polypeptide which can be renatured from a denaturing gel and shown to bind ARS1 DNA. We have designated this protein DBF-A, DNA-binding factor A.

Adenosine Triphosphate↗
PubMed1992

Role of multifunctional autonomously replicating sequence binding factor 1 in the initiation of DNA replication and transcriptional control in Saccharomyces cerevisiae.

Autonomously replicating sequence (ARS) binding factor 1 (ABF1) is an abundant DNA-binding protein that specifically recognizes the motif RTCRYN5ACG at many sites in the yeast genome, including promoter elements, mating-type silencers, and ARSs. Mutational analysis of these sites suggests that ABF1 is involved in constitutive and carbon source-regulated transcriptional activation, transcriptional silencing, and ARS activity. To better assess the role of ABF1 in DNA replication and transcriptional control, temperature-sensitive lethal mutations in the ABF1 gene were isolated. Several of the abf1(Ts) strains show rapid growth arrest at the nonpermissive temperature. At the semipermissive temperature, these strains show an ARS-specific defect in the mitotic stability of ARS-CEN plasmids, such that the abf1 mutants show defects in ARS function identical to those of mutants bearing the mutations in the cis-acting ABF1 binding sites analyzed previously by numerous investigators. Flow cytometric analysis and in vivo DNA labeling experiments on an alpha-factor synchronized abf1(Ts) strain showed that at the nonpermissive temperature, these cells fail to progress efficiently from G1 through S phase and synthesize DNA at 25% of the level seen in the isogenic ABF1 strain. RNA synthesis is also reduced in the abf1(Ts) strains. In addition, transcriptional activation by an ABF1 binding site upstream activation sequence is completely defective in an abf1(Ts) strain at the semipermissive temperature. These phenotypes provide evidence that the same protein, ABF1, functions in the initiation of DNA replication and transcriptional activation.

Alleles↗
PubMed1992

Changes resulting from increasing appointment length: practical and theoretical issues.

The experience of one urban teaching practice in changing its appointment length from 7.5 to 10.0 minutes is described. Observed benefits to patients attending routine surgeries included an increased consultation time (mean 8.6 minutes before, 9.1 minutes after) and reduced waiting time (mean 19.1 minutes compared with 14.6 minutes). Overall, workload was unchanged but improving the 'fit' between supply and demand was associated with loss of flexibility--a greater number of extra patients required to be seen, apparently because fewer appointments were available at the start of each day. Waiting and consultation times in teaching surgeries and trainee surgeries (booked throughout at 10.0 minute intervals) were unchanged in response to the new arrangements. The changes introduced were well received by medical and reception staff although their response was not formally measured. Planning the organization of an appointment system requires several distinct decisions to be made. The preferred or actual average length of consultations has to be decided and booking arrangements designed to enable this to take place without the doctors persistently running over time. The number of appointments per week required to meet anticipated demand has to be calculated on the basis of list size and expected annual consultation rate. However, an exact fit between supply and demand will lead to congestion of the system and it appears that flexibility in the form of an overprovision of appointments to projected demand of about 120% should be built in. Sufficient vacant slots must be provided at the start of each day to allow sufficient flexibility to avoid excessive numbers of patients having to be accommodated.(ABSTRACT TRUNCATED AT 250 WORDS)

Appointments and Schedules↗
PubMed1991

Identification and purification of a factor that binds to the Mlu I cell cycle box of yeast DNA replication genes.

In Saccharomyces cerevisiae, the genes encoding at least 10 enzymes involved in DNA replication are periodically expressed in the late G1 and S phases of the cell cycle. All of these genes have one copy or more of the sequence ACGCGT, which conforms to the recognition site for the Mlu I restriction endonuclease. For the CDC21, CDC9, and POL1 genes, the Mlu I site has been shown to be absolutely required for periodic transcription. Using nuclear extracts fractionated by conventional and oligonucleotide affinity chromatography, we have purified a 17-kDa protein that recognizes the Mlu I motif. Synthetic oligonucleotides containing mutated Mlu I sites do not bind the protein. In contrast, synthetic oligonucleotides derived from the CDC2, CDC6, and CDC21 genes, which are expressed with the same timing as POL1, bind purified protein efficiently.

Bacterial Proteins↗
PubMed1991

A cell cycle-responsive transcriptional control element and a negative control element in the gene encoding DNA polymerase alpha in Saccharomyces cerevisiae.

Transcription of the POL1 gene of Saccharomyces cerevisiae, which encodes DNA polymerase alpha, the DNA polymerase required for the initiation of DNA replication, has previously been shown to be cell cycle regulated. To understand how the POL1 gene senses cell cycle position, we have investigated the cis-acting elements that respond to the factors that govern cell cycle progression. In this report we demonstrate that a region of 54 nucleotides containing the repeated element ACGCGT, which conforms to an Mlu I restriction endonuclease recognition site, contains all information necessary for transcriptional activation and cell cycle responsiveness. Although oligonucleotides lacking either one or both of the repeated Mlu I sites can function as an upstream activating sequence, the presence of at least one Mlu I site stimulates expression and, moreover, is absolutely essential for cell cycle regulation. A synthetic oligonucleotide corresponding to a 19-base-pair sequence in the POL1 promoter containing one Mlu I site can function as an autonomous cell cycle-responsive upstream element (upstream activation sequence) with temporal regulation indistinguishable from that previously described for the POL1 gene. Thus, the Mlu I site is an essential part of a cis-acting element responsible for the observed periodic activation. This sequence differs from previously defined cell cycle-responsive transcriptional control elements in the yeast HO endonuclease and histone genes. We also present evidence for a negative regulatory element in the 5' flanking region of the Mlu I upstream activation sequence.

Base Sequence↗
PubMed1991

The CDC7 protein of Saccharomyces cerevisiae is a phosphoprotein that contains protein kinase activity.

The CDC7 protein of Saccharomyces cerevisiae may be involved in the G1/S-phase transition and/or in the initiation of mitotic DNA synthesis. The CDC7 gene has two in-frame AUG codons as possible translation start sites, which would produce 58- and 56-kDa proteins, respectively. Both p58 and p56 derived from recombinant plasmids complement the temperature-sensitive growth defect of the cdc7-1 allele. To determine the biochemical function of the CDC7 protein, the CDC7 gene was cloned and polyclonal antibodies were produced against the CDC7 protein. CDC7 immune complexes prepared from yeast with these antibodies phosphorylate histone H1. Kinase activity is thermolabile in strains carrying the cdc7-1 temperature-sensitive mutant allele and is elevated greater than 10-fold in strains carrying plasmids overexpressing either p56 or p58, confirming that the kinase in the immunoprecipitates is the CDC7 gene product. In addition, we show that CDC7 is a phosphoprotein itself. Indirect immunofluorescence and biochemical fractionation show that the CDC7 protein is present at relatively high concentrations in the nucleus compared with the cytoplasm, suggesting that nuclear proteins may be substrates for the CDC7 protein.

Cell Cycle↗
PubMed1991

Influence of parents' physical activity levels on activity levels of young children.

To determine the relationship between activity levels of parents and those of their young children, we monitored physical activity with a mechanical device, the Caltrac accelerometer, in one hundred 4- to 7-year-old children and in 99 of their mothers and 92 of their fathers. During 1 year in the Framingham Children's Study, data were obtained for an average of more than 10 hours per day for 8.6 +/- 1.8 days for the children, for 8.3 +/- 2.1 days for their mothers, and for 7.7 +/- 2.3 days for their fathers. Children of active mothers (average Caltrac accelerometer counts per hour greater than the median) were 2.0 times as likely to be active as children of inactive mothers (95% confidence interval = 0.9, 4.5); the relative odds ratio of being active for the children of active fathers was 3.5 (95% confidence interval = 1.5, 8.3). When both parents were active, the children were 5.8 times as likely to be active (95% confidence interval = 1.9, 17.4) as children of two inactive parents. Possible mechanisms for the relationship between parents' and child's activity levels include the parents' serving as role models, sharing of activities by family members, enhancement and support by active parents of their child's participation in physical activity, and genetically transmitted factors that predispose the child to increased levels of physical activity.

Child↗
PubMed1990

E. coli oriC and the dnaA gene promoter are sequestered from dam methyltransferase following the passage of the chromosomal replication fork.

We have examined individual GATC sites throughout the E. coli genome for their kinetics of remethylation by dam methyltransferase following the passage of the chromosomal replication fork. We present evidence for three major conclusions: that oriC is a single function unit that is specifically sequestered from dam methyltransferase for a significant period of time and then released; that the dnaA promoter region is subject to sequestration analogous to that observed at oriC and thus that hemimethylation-dependent sequestration is a general phenomenon; and that each round of replication initiation triggers a transient, temporally coordinate block in both reinitiation at oriC and expression of the dnaA gene. These and other observations are all consistent with the notion that hemimethylation in these two regions acts coordinately to ensure that every origin undergoes initiation once and only once per cell cycle; other possible roles for sequestration at dnaA are also considered.

Bacterial Proteins↗
PubMed1990

Transplantation of pituitary grafts fail to restore immune function and to reconstitute the thymus glands of aged mice.

There is evidence to indicate that the neuroendocrine and immune systems can interact. Thus, neuroendocrine hormones can modulate a variety of immune functions and there have been attempts to manipulate the neuroendocrine system of aged animals to enhance immune function. We have previously shown that the transplantation of a syngeneic pituitary gland under the kidney capsule of young adult mice elevates serum prolactin and enhances immune responsiveness. In the present study pituitary glands were transplanted under the kidney capsule of 22-month-old mice to determine if this maneuver can enhance a number of immunologic parameters. The results demonstrate that aged animals bearing transplanted pituitary grafts for 10 days did not exhibit any enhancement in their primary antibody response to sheep red blood cells, splenic T or B-cell mitogen responsiveness or restoration of thymic architecture. When these immunologic assessments are performed on animals bearing pituitary grafts for 28 days, the IgM and IgG primary antibody responses and splenic T-cell responsiveness are enhanced but repopulation of the thymus still does not occur. Importantly, this enhancement does not restore immunocompetence to levels observed in young mice.

Aging↗
PubMed1990

Purification of DNA polymerase II stimulatory factor I, a yeast single-stranded DNA-binding protein.

Incidental to the purification of yeast DNA polymerase II was the observation that various chromatographic fractions contained activities that stimulated synthesis by this polymerase. In this paper we report the purification and initial characterization of one such factor, stimulatory factor I (SFI). SFI, which is associated with an apparent complex of three polypeptides of 66, 37, and 13.5 kDa, binds preferentially to single-stranded DNA, possibly explaining its ability to stimulate DNA polymerase II. Single-stranded DNA-binding activity is associated with the 66-kDa polypeptide.

Centrifugation, Density Gradient↗