Biomedical subjects
Publications and source records attributed to D M Prescott.
Hypotrichs are a large group of ciliate species that cut, splice, reorder and eliminate DNA sequences to an extraordinary extent during their sexual life cycle. Such DNA processing occurs when a ciliate converts a copy of its germ-line nucleus into a somatic nucleus after cell mating.
The present study investigates the effects of nitroprusside, a potent vasodilating agent, on tissue temperature during local hyperthermia in five normal and five tumor-bearing dogs. Caudal thigh muscles were heated in normal dogs and muscle temperatures were recorded during hyperthermia. Tumor-bearing dogs received two hyperthermia treatments during a course of radiation therapy. Temperatures were recorded in tumor and surrounding normal tissues. Mean arterial pressure was decreased by approximately 40-45% during nitroprusside infusion and was associated with a compensatory increase in heart rate and increases in tissue temperature. In normal dogs, muscle temperatures increased an average of 1.7 degrees C with nitroprusside administration. When nitroprusside was administered at the beginning of local hyperthermia to induce step-down heating, approximately 48% of the measured positions in caudal thigh muscle achieved a temperature greater than or equal to 43 degrees C, sufficient to induce step-down heating, during the hyperthermia episode. In tumor-bearing dogs, there was a significant increase in tumor and normal tissue temperatures during nitroprusside administration. Estimated T90 and T50 descriptors increased by 0.9 degrees C and 1.6 degrees C, respectively, for tumor tissue and by 0.4 degrees C and 1.2 degrees C, respectively, for normal tissue. Despite the increase in normal tissue temperatures no toxicity was observed in these dogs. Nitroprusside may be a useful agent for manipulation of tumor temperatures during the entire hyperthermia treatment or for a short time period at the initiation of treatment to induce step-down heating.
Following cell mating in ciliates, a copy of the micronuclear genome is processed into a new macronucleus through massive cutting, reordering, splicing, elimination, and amplification of the DNA. DNA processing includes the deletion of short interrupting elements called internal eliminated sequences (IESs), followed by the splicing of the remaining segments, known as macronuclear destined sequences (MDSs). The MDSs in some micronuclear genes, such as actin I, are scrambled and must be reordered during IES removal and MDS splicing to yield a functional gene. Here, we describe the cloning, sequencing, and characterization of a different scrambling pattern for the gene that encodes the alpha subunit of the telomere-binding protein of Oxytricha nova. The micronuclear gene is made up of 14 MDSs in the scrambled order 1-3-5-7-9-11-2-4-6-8-10-12-13-14. Only the scrambled version is present in the micronucleus, and only the unscrambled version is present in the macronucleus. We propose that unscrambling occurs by homologous recombination guided by pairs of direct repeats at MDS-IES junctions. The patterned array of scrambling may be a clue to the origin of scrambling in this gene.
The effects of regional hyperthermia (42 degrees C for 70 min) on the antitumor activity of melphalan were examined in athymic mice bearing melphalan-resistant human rhabdomyosarcoma (TE-671 MR) xenografts growing in the right hind limb, and results were compared with similar studies of melphalan-sensitive (TE-671) parent xenografts. Melphalan alone at a dose of 36 mg/m2 (0.5 of the 10% lethal dose) produced growth delays of 4.1 to 10.2 days in TE-671 MR xenografts and 21.8 to 28.7 days in TE-671, respectively. Hyperthermia alone produced growth delays of 0.9 days in TE-671 MR xenografts and 0.8 days in TE-671. Combination therapy with melphalan and hyperthermia produced growth delays of 7.2 to 13.3 days in TE-671 MR xenografts and 34.3 to 42.8 days in TE-671, respectively, representing a mean thermal enhancement ratio of 1.7 in TE-671 MR and 1.5 in TE-671. Measurement of glutathione levels in TE-671 MR xenografts following treatment with melphalan, hyperthermia, or melphalan plus hyperthermia revealed significant reductions in glutathione content with the nadir (60% of control values) seen 6 h following treatment. Glutathione levels in TE-671 xenografts following identical therapy revealed no differences from control values. Hyperthermia plus melphalan did not result in a higher tumor-to-plasma melphalan ratio compared with treatment with melphalan alone in either TE-671 MR or TE-671 xenografts. These studies suggest that heat-induced alterations in tumor glutathione or melphalan levels are not responsible for the increase in melphalan activity produced by hyperthermia. Combination therapy with melphalan plus regional hyperthermia offers promise for treatment of melphalan-resistant neoplasms.
The ends or telomeres of the linear chromosomes of eukaryotes are composed of tandem repeats of short DNA sequences, one strand being rich in guanine (G strand) and the complementary strand in cytosine. Telomere synthesis involves the addition of telomeric repeats to the G strand by telomere terminal transferase (telomerase). Telomeric G-strand DNAs from a variety of organisms adopt compact structures, the most stable of which is explained by the formation of G-quartets. Here we investigate the capacity of the different folded forms of telomeric DNA to serve as primers for the Oxytricha nova telomerase in vitro. Formation of the K(+)-stabilized G-quartet structure in a primer inhibits its use by telomerase. Furthermore, the octanucleotide T4G4, which does not fold, is a better primer than (T4G4)2, which can form a foldback structure. We conclude that telomerase does not require any folding of its DNA primer. Folding of telomeric DNA into G-quartet structures seems to influence the extent of telomere elongation in vitro and might therefore act as a negative regulator of elongation in vivo.
The purpose of this study was to evaluate the effect of hyperthermia on the histologic and functional response of the canine kidney, a late-responding normal tissue, to irradiation. Both kidneys were irradiated. Radiation was delivered in single doses of 0, 10, or 15 Gy. Whole-body hyperthermia was used to produce renal kidney temperatures approximating 42.0 degrees C for 60 min. Thirty-six beagles were placed randomly in the following six treatment groups: control, whole-body hyperthermia alone, 10 Gy alone, 10 Gy + whole-body hyperthermia, 15 Gy alone, and 15 Gy + whole-body hyperthermia. Renal histologic and functional changes were assessed at 1 to 9 months after therapy. No changes were seen in glomerular filtration rate or renal tissue volumes in control or hyperthermia alone groups. Renal vascular and glomerular volumes were not affected significantly by any combination of hyperthermia and/or radiation. In all groups receiving radiation, glomerular filtration rate decreased, percentage renal tubular volume decreased, and interstitial volume increased significantly after therapy. The magnitude of these changes in the functional and histologic response of the kidney and the latent period before expression of this damage were dependent on radiation dose. However, hyperthermia did not modify expression of radiation damage in the kidney based on glomerular filtration rate and histologic quantification of renal tissue components.
An enzymatic activity in crude extracts of macronuclei from the hypotrichous ciliate Oxytricha nova catalyzes the synthesis of RNA consisting of (C4A4)n using an oligodeoxynucleotide template of the telomeric sequence (dG4T4)n. Single-stranded (dG4T4)n is an effective template if it has a random sequence at its 5' end. The enzyme will not use a (dG4T4)n template of any length (up to 64 bases) if it lacks a random sequence at the 5' end. With a random, single-stranded sequence at the 5' end, the (dG4T4)n oligodeoxynucleotide must be at least 36 bases long to work as a template. A 16-base, single-stranded region of (dG4T4)2 is an effective template when joined to a 20-base double-stranded region of (dG4T4)n/(dA4dC4)n, a structural arrangement that is the same as the native telomere of Oxytricha macronuclear DNA. The RNA-synthesizing activity is unaffected by 1.0 mg/ml of alpha-amanitin. Macronuclear extracts have an alpha-amanitin-insensitive, RNA-polymerizing activity that can use a random 55mer oligodeoxynucleotide as a template. This enzyme activity may be the same one that uses (dG4T4)n templates to make (C4A4)n RNA. The (C4A4)n RNA made in the reaction can prime DNA synthesis by the E. coli DNA polymerase I Klenow fragment. Therefore, the RNA polymerase activity fulfills the requirements of the telomere DNA primase that we postulated for replication of telomeres in hypotrichs (Zahler and Prescott, 1988, Nucleic Acids Research 16, 6953-6972).
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During the development of a macronucleus from a micronucleus after cell mating in hypotrichs all the genes (approximately 20,000) are excised from micronuclear chromosomes as individual small DNA molecules. Telomeres are added to the ends of each gene-sized molecule and each is amplified, mostly by approximately 1000-fold, to yield a transcriptionally active macronucleus. As a part of the study of the excision of genes from chromosomes, we have cloned six fragments of chromosomal DNA from Oxytricha nova, each containing a full copy of an actin gene, for comparison with the structure of the actin-encoding DNA molecule in the macronucleus. All six micronuclear actin clones had the same overall organization as judged by restriction mapping. Two micronuclear actin clones were sequenced. These differ from one another at a few nucleotide positions but both prescribe precisely the same actin polypeptide. Both micronuclear actin genes contain nine exons separated by eight intron-like sequences. The macronuclear gene contains these nine exons without intron-like segments. Assigning the order 1 through 9 to the nine micronuclear exons, the order in the macronucleus is 8-7-1-2-4-3-5-9-6. In the micronuclear actin gene, all nine exons possess terminal repeat sequences. These repeat sequences provide precise directions for reordering and joining of the nine exons to yield the exon order in the macronuclear gene. Polymerase chain reaction analysis of micronuclear DNA of the related species, Oxytricha trifallax, shows that the actin gene has an unorthodox arrangement in this species also.
We have found abundant telomere-specific terminal transferase activity in crude macronuclear extracts from vegetatively growing cells of the hypotrichous ciliate Oxytricha nova. This activity adds two to seven tandem repeats of the sequence GGGGTTTT (the Oxytricha telomeric repeat) to the 3' end of oligonucleotide primers ending in repeats of G4T4 and always adds the repeats in the proper phase. The activity requires the presence of micromolar amounts of dGTP and dTTP as well as single-stranded oligomer primers ending 3' with repeats of the Oxytricha telomeric sequence. A nuclease activity is present in the extracts which is closely balanced with telomere terminal transferase activity. We propose a simple model for replication of the ends of linear DNA molecules based on the telomere terminal transferase.
We have cloned and sequenced a 1.6-kb macronuclear molecule encoding actin from the hypotrichous ciliate Oxytricha nova. High-stringency Southern hybridization to native and digested macronuclear DNA shows that there is only one 1.6-kb actin-encoding molecule in O. nova. The 227-nucleotide 5' leader sequence contains AT-rich stretches punctuated by short GC regions. The AT-rich regions contain TATA-like sequences. However, other known eukaryote transcription regulatory sequences were not found. The 249-nucleotide 3' trailer sequence is also AT-rich and does not contain any obvious known eukaryotic mRNA processing signals. Sequence comparison with a closely related species, O. fallax, shows an 87% sequence similarity in the coding regions and an almost total lack of similarity in the noncoding regions of the molecules. However, a few small sequence similarities and motifs appear in the noncoding regions of the actin-encoding molecules of these two species. The actin-encoding molecule of O. nova could encode a polypeptide 374 amino acids long, the same size as some vertebrate cytoplasmic actins. Contrary to a previous report, we show that the actin-encoding molecule of O. fallax also codes for a polypeptide 374 amino acids long.
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A convenient method for double-label autoradiography is described that uses an aqueous mountant, Gelutol (polyvinyl alcohol), which keeps the gelatin spacer in the final autoradiograph permanently swollen to a thickness of around 18 microns in contrast to its 5 microns thickness during exposure of the autoradiograph. This greatly improves optical discrimination between upper and lower layers without the loss of sensitivity or resolution that would result if a 18 microns spacer were used during exposure.
Cells of the V79-8 line grow with little or no G1 period. Serum or isoleucine deprivation results in cell death rather than entry into quiescence. Introduction of a G1 period of 4-5 h by treatment with a mutagen is not sufficient to re-establish the ability to enter quiescence. The experiments described here show that introducing a G1 period by mutation that lowers the rate of protein synthesis does not restore to the cell any competence to arrest in G1 and enter a state of quiescence.
An isoleucine arrest point in G1 was determined by two methods for CHO and 3T3 cells. In the first method the fraction of cells entering S after isoleucine deprivation was assessed by [3H]thymidine labelling and autoradiography. In the second method cells entering S after isoleucine deprivation were identified by double-label autoradiography using [3H] and [14C]thymidine. From the fraction of cells entering S, determined by the two methods, the arrest point in G1 (and entry into G0) is located within the last 40 min of G1.
To examine the hypothesis that p53 protein may play a central role in regulating reproduction of mammalian cells, we compared the absolute amounts and relative rates of synthesis of p53 protein in two pseudonormal cell lines, 3T3 and C3H 10T1/2, during quiescence, during log proliferation, and in quiescent cells stimulated with serum. The absolute amount of p53 protein per cell was found to be severalfold lower in quiescent cells than in log-phase cells. The ratio of the rate of synthesis of p53 protein to the rate of synthesis of total protein was slightly higher in quiescent cells than the same ratio in log-phase cells. Thus, entry into quiescence is not accompanied by a differential switch-off of synthesis of p53 protein. In quiescent cells stimulated with serum the amount of p53 protein per cell and its rate of synthesis increase, but only in proportion to the increase in total protein per cell and the increase in rate of total protein synthesis. Similarly, 12-14 h after serum stimulation, the time of the G1 to S transition, the accumulated increase in p53 protein per cell is about what would be expected for a short-lived protein whose rate of synthesis has increased in proportion to the increase in rate of synthesis of total protein. The results are not those expected for a protein that functions specifically in release from quiescence or in transition from G1 to S.
Three gene-sized molecules cloned intact from macronuclear DNA served as hybridization probes to study excision of these molecules from chromosomes and their processing during macronuclear development in the hypotrich Euplotes crassus. These molecules occur in integrated forms within polytene chromosomal DNA during macronuclear developmental. After transection of the polytene chromosomes, the three molecules occur in intermediate forms. One of the three molecules first appeared in a large intermediate that was subsequently replaced by a second intermediate, approximately 140 bp larger than the final molecule. The other two macronuclear molecules were detected only in intermediates approximately 140 bp larger than the mature form. These penultimate intermediates are larger by virtue of oversized telomeres, which are pared to yield the mature gene-sized molecules.