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Biomedical subjects

G F Morris

Publications and source records attributed to G F Morris.

48 records · Page 3Linked to original sources
PubMed1990

Analysis of the proliferating cell nuclear antigen promoter and its response to adenovirus early region 1.

The levels of the mRNA for the proliferating cell nuclear antigen (PCNA), a DNA replication factor, increase upon growth stimulation of quiescent cells. To study the transcriptional aspect of this response, we have cloned a PCNA gene fragment from size-fractionated human placental DNA. This fragment contains 1269 nucleotides upstream from the PCNA transcriptional start site and includes an Alu sequence that is transcribed in vitro. The PCNA genomic DNA promotes transcription of a linked heterologous reporter gene in HeLa and 293 cells. Transient expression assays and in vitro transcription analyses showed that 249 nucleotides of upstream sequence are sufficient for full promoter activity in HeLa cells, whereas only 172 nucleotides are needed in 293 cells. Co-transfection with a plasmid expressing the adenovirus E1 gene transactivates the PCNA promoter in HeLa cells. An E1-responsive element maps in the 85-nucleotide region immediately upstream of the site of transcription initiation.

Adenovirus Early Proteins↗
PubMed1989

Regulation of proliferating cell nuclear antigen during the cell cycle.

The proliferating cell nuclear antigen (PCNA), also known as cyclin and DNA polymerase delta auxiliary factor, is present in reduced amounts in nongrowing cells and is synthesized at a greater rate in the S phase of growing cells. The recently discovered involvement of PCNA in DNA replication suggested that this pattern of expression functions to regulate DNA synthesis. We have investigated this possibility further by examining the synthesis, stability, and accumulation of PCNA in HeLa cells fractionated by centrifugal elutriation into nearly synchronous populations of cells at various positions in the cell cycle. In these fractionated cells we found that there is an increase in the rate of PCNA synthesis with a peak in early S phase of the cell cycle, but the magnitude of the increase is only 2-3-fold. This change reflects similar changes in the amount of PCNA mRNA. The fluctuating synthesis of PCNA maintains this protein at a roughly constant proportion of the total cell protein, although the amount doubles/cell in the cell cycle. Consistent with this observation, the stability of PCNA does not differ significantly from that of total cellular protein in synchronized HeLa cells. We also observed that a maximum of one-third of the total PCNA is tightly associated with the nucleus, presumably in replication complexes, at the peak of S phase. We conclude that the cyclic synthesis of PCNA in cycling HeLa cells maintains PCNA in excess of the amount involved directly in DNA replication and the amount of the protein neither fluctuates significantly with the cell cycle nor is limiting for DNA synthesis.

Antigens, Neoplasm↗
PubMed1989

Sea urchin early and late H4 histone genes bind a specific transcription factor in a stable preinitiation complex.

Early embryonic H4 (EH4) and H2B (EH2B) and late embryonic H4 (LH4) histone genes were transcribed in vitro in a nuclear extract from hatching blastula embryos of the sea urchin Strongylocentrotus purpuratus. The extract was prepared by slight modifications of the methods of Morris et al. (G. F. Morris, D. H. Price, and W. F. Marzluff, Proc. Natl. Acad. Sci. USA 83:3674-3678, 1986) that have been used to obtain a cell-free transcription system from embryos of the sea urchin Lytechinus variegatus. Achievement of maximum levels of transcription of the EH4 and LH4 genes required a 5- to 10-min preincubation of template with extract in the absence of ribonucleoside triphosphates. This preincubation allowed the formation of a stable complex which was preferentially transcribed compared with a second EH4 or LH4 template that was added 10 min later. Although the EH4 gene inhibited both EH4 and LH4 gene transcription in this assay and although the LH4 gene inhibited both EH4 and LH4 genes, neither of these genes inhibited transcription of the EH2B gene. Preincubation with the EH2B gene had no effect on the transcription of subsequently added EH4 or LH4 genes. Using this template commitment assay, we showed that the site of binding of at least one essential factor required for transcription of both EH4 and LH4 genes was located between positions -102 and -436 relative to the 5' terminus of the EH4 mRNA. Moreover, deletion of this region resulted in a reduction in EH4 gene transcription in vitro. The sea urchin gene-specific trans-acting factors, in the analysis of the cis-acting sequences with which they interact, and in biochemical studies on the formation of stable transcription complexes.

Animals↗
PubMed1986

Synthesis of U1 RNA in a DNA-dependent system from sea urchin embryos.

A soluble extract prepared from blastula nuclei of sea urchin (Lytechinus variegatus) embryos accurately transcribes cloned sea urchin DNA. This extract synthesizes U1 RNA using a cloned U1 RNA gene as a template. The U1 RNA is initiated accurately, and a portion of the transcripts has the correct 3' end as judged by gel electrophoresis. Longer transcripts also are formed that extend at least 280 bases 3' of the gene, and some extend as far as 800 bases 3' of the gene. A template containing 203 bases 5' of the gene gave as efficient transcription as did the whole U1 gene. Accurate 3' end formation was obtained with a template extending only 34 bases 3' of the gene, but efficient 3' end formation required sequences between 34 and 67 bases 3' of the gene.

Animals↗
PubMed1985

Structure of the sea urchin U1 RNA repeat.

The genes coding for U1 RNA in the sea urchin L. variegatus are present in a 1400 base pair tandem repeat. One member of the repeat has been cloned and its sequence determined. The repeat unit contains a single copy of the gene for L. variegatus U1 RNA. This gene encodes an RNA which is 75% homologous to mammalian U1 RNA. The L. variegatus U1 RNA could assume a secondary structure similar to that proposed for other U1 RNAs. In addition the L. variegatus U1 RNA is precipitated by anti-SM and anti-RNP antisera. Analysis of the L. variegatus genomic DNA using the cloned U1 gene as a probe reveals a major and a minor type of repeat unit. The two repeated units are the same length but differ in a number of restriction enzyme sites clustered 200-500 bases down-stream from the gene. The monomer we have cloned and sequenced is a representative of the minor repeat. A sequence (GATAA) which is -41 to -37 bases 5' to the gene has homology to the putative RNA polymerase II promoter. Fifteen bases 3' of the gene is a sequence (CAAAGAAAGAAAA) which is very similar to the sequence found 3' of the sea urchin histone genes. The two Hha I, Hpa II and Ava I sites in the repeat are all unmethylated in sperm DNA.

Animals↗
PubMed1985

Synthesis of U1 RNA in isolated nuclei from sea urchin embryos: U1 RNA is initiated at the first nucleotide of the RNA.

Nuclei from sea urchin blastula embryos synthesize a variety of small RNAs, one of which has identical mobility with sea urchin U1 RNA. This RNA is synthesized by RNA polymerase II and, in a hybridization-selection experiment, was selected by the cloned sea urchin U1 gene. The U1 RNA was initiated with ATP, but not GTP, in isolated nuclei with beta-S- and gamma-S-ribonucleotide triphosphates as substrates. The U1 RNA containing thiophosphate at the 5' end was not capped but accumulated as an uncapped transcript from which the thiophosphate could be removed with calf intestinal phosphatase.

Animals↗
PubMed1983

A factor in sea urchin eggs inhibits transcription in isolated nuclei by sea urchin RNA polymerase III.

Isolated nuclei from sea urchin embryos synthesize RNA at a rate comparable to other animal cell nuclei. All three RNA polymerases are active as judged by alpha-amanitin sensitivity and hybridization to specific cloned DNAs. Extracts were prepared from sea urchin eggs and embryos by extraction with 0.35 M KCl. None of the crude extracts had a large effect on total RNA synthesis. However, extracts from sea urchin eggs inhibited RNA polymerase III activity in nuclei from blastula and gastrula embryos. There was no effect on the synthesis of ribosomal RNA by RNA polymerase I or on the synthesis of two RNA polymerase II products, histone mRNA and the sea urchin analogue of U1 RNA. The inhibitor is present in two different species of sea urchin and has been 50-fold purified by diethylaminoethylcellulose and hydroxylapatite chromatography. The inhibitor is not present in extracts prepared from sea urchin blastula embryos.

Animals↗
PubMed1982

Sea urchin small nuclear RNA genes are organized in distinct tandemly repeating units.

The genes coding for the two major small nuclear RNAs in the sea urchin are organized in independent tandem repeating units. The small nuclear RNAs, N1 and N2 were purified from gastrula embryos of Lytechinus variegatus. These RNAs are analogous to the U series of RNA in mammalian cells as judged by their identical 5' termini and the sequence homology of the N1 urchin RNA and U1 mouse RNA. These RNAs were polyadenylated with E. Coli adenylate transferase. A 32PO4 labeled copy of each RNA was made with RNA-dependent DNA polymerase. This copy was used to probe the gene organization of these RNAs by hybridizing to restriction enzyme digests of sperm DNA. Each of these RNAs is coded in a tandemly repeated cluster (at least 30 kb) with a repeat length of 1100-1400 bases. The N1 and N2 clusters are distinct. The N1 repeat has been cloned and the repeating organization confirmed with the cloned gene.

Animals↗
PubMedDate not supplied

The parasacral sciatic nerve block.

BACKGROUND AND OBJECTIVES: The clinical utility of a new parasacral approach for conduction block of the sciatic nerve was investigated, with critical examination of onset, extent, and success rates when this block was used for surgical procedures below the knee. METHODS: Thirty ASA I-III patients presenting for surgery on the lower limb were enrolled. All received 30 mL of 1.5% lidocaine with 1:200,000 epinephrine following nerve stimulator identification of the sciatic nerve at < or =0.2 mA or less. Trans-sartorial saphenous nerve blocks were performed to provide anesthesia to the medial leg. RESULTS: Overall success for surgical anesthesia with this block was 97%. All components of the sacral plexus could be blocked with this approach, and 93% of patients displayed evidence of obturator nerve motor block. However, no patient displayed evidence of obturator sensory anesthesia that could be mapped. Saphenous nerve blocks were 100% effective in providing surgical anesthesia for the procedures performed. CONCLUSIONS: The parasacral approach to the sciatic nerve exhibits a high success rate, resulting in anesthesia of the entire sacral plexus and generally in motor block of the obturator nerve was an interesting observation.

Adult↗
PubMedDate not supplied

Continuous parasacral sciatic nerve block: two case reports.

OBJECTIVE: This study investigated the use of a continuous parasacral sciatic nerve block for anesthesia and postoperative analgesia for lower extremity surgery. METHODS: A continuous parasacral sciatic nerve block was performed in two patients (triple ankle arthrodesis and below-knee amputation). The sacral plexus was identified using an insulated Tuohy needle and a nerve stimulator. A catheter was placed near the elements of the sacral plexus via the Tuohy needle. RESULTS: In both patients, surgical anesthesia was successfully established through the parasacral catheter with lidocaine 1% (1/200,000 epinephrine), and postoperative analgesia was successfully established with a bolus of bupivacaine 0.375% (1/200,000 epinephrine) and maintained with a continuous infusion of bupivacaine 0.1% (8 mL/h) for 48 hours. CONCLUSION: We conclude that continuous parasacral sciatic nerve block can provide anesthesia and long-term analgesia for operative procedures of the foot and leg.

Amputation, Surgical↗